Enrichment of nucleic acid targets

ABSTRACT

Methods and apparatus providing for the isolation of an unknown mutation from a sample comprising wild type nucleic acids and mutated nucleic acids through the application of time-varying driving fields and periodically varying mobility-altering fields to the sample within in an affinity matrix.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalNo. 62/242,704, filed Oct. 16, 2015, which is incorporated by referencein its entirety.

This application is a continuation-in-part of U.S. application Ser. No.14/690,934, filed Apr. 20, 2015, which is a continuation of U.S. Ser.No. 14/021,697, filed Sep. 9, 2013, which is a continuation-in-part ofU.S. application Ser. No. 13/593,143, filed Aug. 23, 2012, which is acontinuation-in-part of U.S. application Ser. No. 13/360,640, filed Jan.27, 2012, which is a continuation of U.S. application Ser. No.10/597,307, filed Sep. 5, 2007 which is a 371 of InternationalApplication No. PCT/CA2005/000124, filed Feb. 2, 2005, which claims thebenefit of U.S. application No. 60/540,352, filed Feb. 2, 2004, and U.S.application No. 60/634,604, filed Dec. 10, 2004. U.S. Ser. No.13/593,143 is additionally a continuation-in-part of U.S. applicationSer. No. 13/153,185, filed Jun. 3, 2011, which claims the benefit ofU.S. application Ser. No. 61/488,585, filed May 20, 2011.

This Application is also continuation-in-part of U.S. application Ser.No. 14/883,234, filed Oct. 14, 2015, which is a continuation of U.S.application Ser. No. 13/739,337 filed Jan. 11, 2013, which claimspriority to U.S. Provisional Application No. 61/586,727 filed Jan. 13,2012 and U.S. Provisional Application No. 61/598,236 filed Feb. 13,2012. All of the aforementioned applications are hereby incorporated byreference in their entireties.

TECHNICAL FIELD

The invention relates to methods and apparatus for moving,concentrating, and enriching particles. The invention has application,for example, in moving, concentrating, or enriching a wide range ofbiological molecules, such as nucleic acids, proteins,bio-macromolecules, and organic or inorganic ions.

BACKGROUND

Standard nucleic acid separation techniques limit researchers' abilitiesto analyze samples for nucleic acids that are present in low abundance,such as mutations. In particular, it is difficult to resolve rarenucleic acids which are present at low concentrations in the presence ofclosely-related nucleic acids, e.g., wild-type DNA. To overcome thisproblem, typically all of the nucleic acids in a sample are amplifiedprior to isolation and analysis. For example, using Polymerase ChainReaction (PCR) amplification, each nucleic acid can be amplified onemillion times (or more). Theoretically, there will be a million-foldincrease of each nucleic acid originally present, and, thus, a greateropportunity to isolate and find the nucleic acids in low abundance.

In practice, however, PCR amplification has significant drawbacks whenit is used to analyze nucleic acids that are present in low abundance.The PCR reaction is stochastic and to the extent that a low-abundancenucleic acid is not amplified in the first few rounds of PCR, it likelywill not be detected. In addition, PCR amplification introduces sequenceerrors in the amplicons. If the error rate is high enough, there can bea significant effect on the resulting sequence data, especially inapplications requiring the detection of rare sequence variants.

Unfortunately, a viable alternative to sequencing plus amplificationdoes not exist. Commonly-available separation techniques do not have theresolution or fidelity to pull enough low-abundance nucleic acids from abackground to be useful. In fact, most separation methods require anamplification step, either before or after separation, to recover enoughlow-abundance nucleic acid for further analysis, e.g. sequencing.

For many years, nucleic acids have been separated from each other usingelectrophoresis. Electrophoresis involves directing the movement ofcharged particles in a medium, such as a gel or liquid solution byapplying an electric field across the medium. The electric field may begenerated by applying a potential across electrodes that are placed incontact with the medium such that electric current can be conducted intothe medium. The movement of the particles in the medium is affected bythe magnitude and direction of the electric field, the electrophoreticmobility of the particles and the mechanical properties, such asviscosity, of the medium. Through electrophoresis, particles that aredistributed in a medium can be transported through the medium.Electrophoresis is commonly used to transport nucleic acids (such as DNAor RNA) through gel substrates. Because different species have differentelectrophoretic mobilities, electrophoresis may be used to separatedifferent species from one another. Conventional electrophoresistechniques are largely limited in application to the linear separationof charged particles. Using conventional electrophoresis techniques, adirect current (DC) electric field or an alternating pulsed-fieldelectrophoretic (PFGE) field is typically applied to a medium so thatparticles in the medium are transported toward an electrode.

Electrophoresis may be used to transport fragments of DNA or othermicroscopic electrically charged particles. Various electrophoresismethods are described in Slater, G. W. et al. Electrophoresis 2000, 21,3873-3887. Electrophoretic particle transport is typically performed inone dimension by applying a direct current (DC) electric field betweenelectrodes on either side of a suitable electrophoresis gel. Theelectric field causes electrically charged particles in the gel to movetoward one of the electrodes. Because the particles experience differentmobilities through the gel due to the DC field, the particles can beseparated. In an alternate application, an asymmetric alternatingcurrent (AC) waveform can cause net drift of electrophoretic particlesdue to nonlinearity of the relationship between particle speed andapplied electric field. This effect can be used to cause particles tomove in one dimension as described in Chacron, M. J., et al. Phys. Rev.E 1997, 56, 3446-3450; Frumin, L. L, et al. Phys. Chem. Commun. 2000,11; and, Frumin, L. L. et al. Phys. Rev. E 2001, 64, 021902.Additionally, Pohl, H. A., Dielectrophoresis: The Behavior of NeutralMatter in Nonuniform Electric Fields Cambridge University Press,Cambridge, UK 1978; Asbury, C. L., et al., Electrophoresis 2002, 23,2658-2666; and Asbury, C. L., et al. Biophys. J. 1998, 74, 1024-1030discloses that dielectrophoresis can be applied to concentrate DNA intwo or more dimensions. However, applications of dielectrophoresis haverequired undesirably high electric field gradients, resulting inbreakdown of the separation media or the samples. Each of thesereferences is incorporated herein in its entirety.

Electrophoresis can also be used to concentrate particles in aparticular location. A problem that can interfere with the successfuluse of electrophoresis for concentrating particles is that there must bean electrode at the location where the particles are to be concentrated.Electrochemical interactions between the electrodes and particles candegrade certain kinds of the particles. For example, where the particlescomprise DNA, the DNA can be damaged by electrochemical interactions atthe electrodes. Additionally, electric fields present duringconventional direct current electrophoresis are divergence-freeeverywhere except at electrodes which can source or sink electriccurrent. Thus, electrophoresis is typically applied in cases whereparticles are caused to move toward an electrode. Once concentrated, theparticles can be obtained by cutting out the portion of the medium inwhich the particles have been concentrated. The particles can then beseparated from the medium by using various purification techniques.

Additional methods of nucleic acid separation are known. References thatdescribe methods for DNA separation include: Slater et al. The theory ofDNA separation by capillary electrophoresis Current Opinion inBiotechnology 2003 14:58-64; Slater et al. U.S. Pat. No. 6,146,511issued 14 Nov. 2000; Frunin et al. Nonlinear focusing of DNAmacromolecules Phys. Rev. E 64:021902; Griess et al. Cyclic capillaryelectrophoresis Electrophoresis 2002, 23, 2610-2617 Wiley-VCH VerlagGmbH & Co. Weinheim (2002). References which describe the use of fieldsto separate particles include: Bader et al. U.S. Pat. No. 5,938,904issued on Aug. 17, 1999; Bader et al. U.S. Pat. No. 6,193,866 issued on27 Feb. 2001; Tessier et al: Strategies for the separation ofpolyelectrolytes based on non-linear dynamics and entropic ratchets in asimple microfluidic device Appl. Phys. A 75, 285-291 (2002); Chacron etal. Particle trapping and self-focusing in temporally asymmetricratchets with strong field gradients Phys. Rev. B 56:3 3446-3550(September 1997); Dean et al. Fluctuation driven ratchets: molecularmotors Phys. Rev. Lett. 72:11 1766-1769 (14 Mar. 1994); Bier et al.Biasing Brownian motion in different directions in a 3-state fluctuatingpotential and an application for the separation of small particles Phys.Rev. Lett. 76:22 4277-4280 (27 May 1996); Magnasco, Forced thermalratchets Phys. Rev. Lett. 71:10 1477-1481 (6 Sep. 1993). Each of thesereferences is incorporated herein in its entirety.

There is a need for techniques that provide for high fidelity enrichmentof nucleic acids without introducing errors into the sample and with anability to isolate rare nucleic acids in a sample and to resolve nucleicacids having similar sequences.

SUMMARY OF THE INVENTION

The invention provides improved methods for recovering nucleic acidsfrom a sample by enriching the sample for a desired nucleic acidspecies, rather than removing those species directly from the sample.Methods of the invention are fundamentally different from conventionalseparation techniques in that methods of the invention create asubsample in which a target that was present in only a small amount inthe original sample becomes the dominant nucleic acid species in thesubsample. For example, a dominant background nucleic acid species maybe driven from a sample into a waste well, leaving behind only targetnucleic acids. As a result, a low-abundance species, for example anucleic acid target that would be difficult to detect using conventionaltechniques that are biased toward the predominant species, is readilydetected using methods of the invention.

Methods of the invention make it possible to separate closely-relatednucleic acid targets, which may differ in concentration by orders ofmagnitude, without substantial loss of the nucleic acid, the detectionof which is desired. Accordingly, the invention provides many of theadvantages of conventional nucleic acid separation and amplificationtechniques (high specificity, high sensitivity, and high speed) withoutthe drawbacks of those techniques (sample loss, introduction of errors,high cost). Methods of the invention are useful to isolate a targetnucleic acid (i.e., a mutation) from a non-target nucleic acid (i.e., awild-type) when the target is present in the original sample at a muchlower concentration than the non-target. Certain specific,disease-indicating, mutations, such as those to oncogenes includingBRAF, KRAS, or EGFR, are known to cause abnormal gene function and canlead to diseases such as cancer. Because mutations can rarely alter agenes function without completely disrupting it, many of these mutationsare have been identified and well characterized which can helpfacilitate their detection.

In other cases, genes such as tumor suppressors, including TP53 and APC,can cause disease simply by ceasing to function. Because any number ofunknown mutations can cause a gene to lose functionality, isolation andanalysis of unknown mutations, especially in certain target genes, canalso prove valuable in disease detection and treatment. In certaininstances, the invention provides a method for enriching nucleic acidswith an unknown mutation over wild type nucleic acids, enablingidentification and analysis of previously unknown mutations. Usingmethods of the invention, it is possible to enrich a sample for a targetnucleic acid 1000, 10,000, 100,000 or even 1,000,000-fold. This fidelityallows the target nucleic acid to be directly sequenced using nextgeneration sequencing. Alternatively, the target nucleic acid can beamplified after enrichment, prior to further processing, e.g.,sequencing. In this case, because the target starting material has beenisolated from the massive excess of non-target nucleic acids, there ismuch less concern for PCR errors in non-target nucleic acids giving riseto phantom target nucleic acids.

In one instance, the invention provides a method to enrich a sample froma target nucleic acid to non-target nucleic acid ratio of X to a targetnucleic acid to non-target nucleic acid ratio of Y, where Y is at least10 times greater than X. The method includes obtaining a sampleincluding a non-target nucleic acid and a target nucleic acid, andenriching the sample for the target. In some examples, the enrichmentincludes applying one or more periodic fields to the sample. The methodcan be achieved even if the target and the non-target differ by only asingle base, and the method does not generate any new molecules duringthe enriching step. Of course, the target and the non-target nucleicacids can differ by more than a single base, for example 20 or fewerbases. In other instances, the target and non-target nucleic adds willdiffer by their methylation, acetylation, or other chemical modificationpatterns. The target and non-target nucleic acids having differingmethylation or acetylation patterns may have the same sequence or theycan have different sequences.

Methods of the invention are used to enrich samples for low-abundancenucleic acids that are important in early diagnosis. The inventionallows resolution of different nucleic acids without amplification inthe isolating step and without regard for the sequence differencebetween a nucleic acid and a variant of it. Accordingly, use of theinvention allows detection and analysis of nucleic acids present in lowabundance in biological samples, such as tissue biopsies, ornon-invasive samples, such as blood or urine. The ability to interrogatelow-abundance nucleic acids is especially important in cancerdiagnostics, where early detection enables effective treatment. Forexample, identification of the presence of a specific mutation maysuggest a particular treatment regimen (e.g., surgery versus radiationtherapy) or suggest that a first line treatment is likely to beineffective, (e.g., the cancer is resistant to docetaxel). Additionally,when mutational events are detected earlier, patients typically havemore options for treatment, as well as the time to identify a preferredtreatment provider.

Methods of the invention are useful in any sample. Preferred samples arederived from tissue or body fluid, for example, tissues, blood, sputum,sweat, urine, tears, feces, aspirates, or a combination thereof.Typically, the biological sample will be from a human, however themethods of the invention may be used to recover nucleic acids from manyorganisms, including, mammals. Moreover, methods of the invention areuseful for the isolation, detection, and interrogation of proteins, aswill be evident from consideration of the detailed description below.

Once a sample is enriched for a target, it will typically be useful toidentify the target using sequencing, hybrid capture, antibodies orother known techniques. Once the target nucleic acid is identified, itwill be possible to correlate its presence in the sample with acondition, or a likely outcome for the subject from which the sample wastaken. For example, the presence of the target nucleic acid may beindicative of a genetic disorder or cancer. Additionally, because themethods of the invention can be used to enrich a sample for multipletargets (serially or in parallel), the invention lends itself todiagnosing diseases by identifying specific biomarker panels thatcorrelate with specific diseases. In some instances the invention willallow the identification of 5 or more targets, e.g., 10 or more targets,e.g., 20 or more targets, e.g., 50 or more targets, e.g., 100 or moretargets. Furthermore, when screening panels comprising multiplebiomarkers are used, the confidence in the resulting diagnosis isincreased. That is, a diagnosis based upon identifying one targetnucleic acid may be the result of noise or error, but when a diagnosisis based upon identifying 10 or more targets simultaneously, it is verylikely not the result of noise or error.

In some cases, the target nucleic acid will be present in low-abundancewith respect to the corresponding wild-type species, and enrichment mayresult in increasing the ratio of target to non-target nucleic acids byat least 100 times or at least 1000 times. In some instances, nucleicacids to be analyzed will be “short” nucleic acids, e.g., having 500 orfewer bases, e.g., having 200 or fewer bases, e.g., having 100 or fewerbases, e.g., having 50 or fewer bases, e.g., having 30 or fewer bases.

In another instance, the invention provides a method for enriching asample, including obtaining a sample having a non-target nucleic acidand a target nucleic acid, wherein the target to non-target ratio in thesample is X, and applying a periodic field to the sample in order togenerate, independent of non-target or target size, a subsample having atarget to non-target ratio of Y, wherein Y is greater than X. In thisinstance no new molecules are generated in the sample during theapplying step.

In another instance, the invention provides a method for enriching asample for a nucleic acid, including obtaining a sample comprising afirst nucleic acid and a second nucleic acid, wherein the second nucleicacid to first nucleic acid ratio in the sample is X, and applying aperiodic field to the sample in order to generate, independent ofnucleic acid size, a subsample having a second nucleic acid to firstnucleic acid ratio of Y, wherein Y is at least 10 times greater than X.In this instance no new molecules are generated in the sample during theapplying step. In this instance, the first and second nucleic acids maybe from a mammal, e.g., a human. Additionally, because of theselectivity of the method it is possible to enrich the sample for asecond nucleic acid that originates from a second mammal, while thesample was recovered from a first mammal. For example, the second mammalmay be a fetus and the first mammal the mother of the fetus, or thesecond mammal may be an assailant and the first mammal a victim oranother individual. In a specific example, the sample may be maternalblood and the first nucleic acid is a maternal nucleic acid and thesecond nucleic acid is a fetal nucleic acid.

Other aspects of the invention provide methods for causing motion ofparticles in a medium. Those methods are useful for concentratingparticles and/or for separating particles of different types from oneanother. Such methods comprise applying a time-varying driving field tothe particles. The driving field applies a time-varying driving forcealternating in direction to the particles. The methods also compriseapplying a mobility-varying field to the particles. The mobility-varyingfield is one or both of: different in type from the driving field, andnon-aligned with the driving field. The driving field andmobility-varying field are applied simultaneously during a period andthe mobility-varying field causes a mobility of the particles in themedium to be time dependent during the period, in a manner having anon-zero correlation with the driving field over the period. Thesemethods may be called SCODA methods.

Another aspect of the invention provides methods and apparatus forextracting charged particles from a medium. Those methods are useful forextracting particles from a medium in which the particles have beenconcentrated by SCODA and may also be applied to extracting from amedium particles that have not been concentrated by SCODA. A buffer inan extraction reservoir is placed to abut a medium containing theparticles to be extracted at a buffer-gel interface. Electrodes areprovided on each side of the buffer-gel interface. By applying a pulsedvoltage potential to the electrodes (wherein the time-averaged electricfield is zero), zero-integrated-field electrophoresis (ZIFE) is appliedto the buffer-gel interface to direct the particles in the gel into theextraction reservoir, where the particles are collected andconcentrated.

Methods of the invention may be used to isolate a second molecule from asample including both the second molecule and a first molecule throughthe application of a time-varying driving field and a periodicallyvarying, mobility-altering field to an affinity matrix comprisingimmobilized probes with a first affinity toward the first molecule thatis greater than a second affinity for a second molecule.

Another method disclosed comprises placing a buffer extraction reservoirnext to a gel solution containing the charged particles to be extracted;applying ZIFE to the buffer-gel interface to direct the particles intothe extraction reservoir; and collecting and concentrating the particlesin the extraction reservoir. A pipette or other device may then be usedto suction the particles from the extraction reservoir. In someembodiments of the invention, the apparatus comprises a gel boat holdinga gel that contains the charged particles to be extracted. A capillarycontaining a small amount of buffer is inserted into the gel solution. Apipette or other device is provided in the capillary for suctioning theparticles that have collected in the buffer. Electrodes are provided oneach side of the buffer-gel interface for generating an electric field.

Further aspects of the invention and features of specific embodiments ofthe invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1I are examples of possible waveforms for driving andmobility-modifying fields.

FIG. 2 is a plot showing a numerical simulation of the path of aparticle.

FIGS. 3A, 3B, 3C, 3D and 3E are schematic diagrams of apparatus that maybe used to practice embodiments of the invention.

FIG. 4A is an example plot of measured DNA velocity as a function ofapplied electric field.

FIG. 4B is a plot illustrating time averaged particle velocity in anapparatus like that of FIG. 3A as a function of radial distance from theorigin.

FIG. 4C is a plot showing the measured DNA spot distance from the originas a function of time.

FIG. 5 shows estimated particle velocity as a function of electric fieldstrength for three molecules in a sieving matrix comprising covalentlybound oligonucleotides.

FIG. 6 is a schematic diagram of apparatus that may be used to explorescodaphoresis using an electric driving field and a thermalmobility-varying field.

FIG. 7A is a schematic diagram of apparatus that may be used to explorescodaphoresis using an electric driving field and an opticalmobility-varying field.

FIG. 7B shows waveforms from the apparatus of FIG. 7A.

FIGS. 8A, 8B, 8C, and 8D show optical mask patterns that may be used tocause concentration of particles at an array of spots.

FIG. 8E shows an array of spots at which particles can be concentratedusing the masks of FIGS. 8A through 8D.

FIG. 9 is a schematic view of apparatus for producing cyclic variationsin viscosity of a medium.

FIGS. 10A, 10B, 10C, and 10D show schematically apparatus for usingmagnetic fields to alter the mobility of particles.

FIG. 11 is a graphical illustration of an exemplary electric field pulseused in ZIFE.

FIG. 12A is a cross-sectional elevation view of an extraction apparatusin accordance with a particular embodiment of the present invention,illustrating molecules of DNA in a solution prior to extraction.

FIG. 12B is a cross-sectional elevation view of the apparatus of FIG.12A, illustrating molecules of DNA extracted from a solution andconcentrated in a small amount of buffer.

FIG. 12C is a plan view of an extraction apparatus similar to that shownin FIG. 12A.

FIG. 13 shows a glass capillary in an extraction experiment using theapparatus and method in accordance with a particular embodiment of theinvention.

FIG. 14 is a graph illustrating the DNA fragment velocity during anexperiment as a function of fragment length and cycle times.

FIG. 15 shows a comparison between a DNA fragment mix and the fragmentdistribution of the same mix, after extraction.

FIG. 16 shows a plot of equation (36) near the duplex meltingtemperature, T_(m), illustrating the relative change in mobility as afunction of temperature.

FIG. 17 shows a plot of mobility versus temperature for two differentmolecules with different binding energies to immobilized probemolecules. The mobility of the high binding energy target is shown bythe curve on the right, while the mobility of the low binding energytarget is shown by the curve on the left.

FIG. 18 shows the effect of an applied DC washing bias on molecules withtwo different binding energies. The solid curve represents the driftvelocity of a target molecule with a lower binding energy to the boundprobes than the molecules represented by the dashed curve.

FIG. 19 shows an example of an electric field pattern suitable for twodimensional SCODA based concentration in some embodiments. Voltagesapplied at electrodes A, B, C, and D, are −V, 0, 0, and 0 respectively.Arrows represent the velocity of a negatively charged analyte moleculesuch as DNA. Color intensity represents electric field strength.

FIG. 20 shows stepwise rotation of the electric field leading tofocusing of molecules whose mobility increases with temperature in oneembodiment of affinity SCODA. A particle path is shown by the arrows.

FIG. 21 shows the gel geometry including boundary conditions and bulkgel properties used for electrothermal modeling.

FIG. 22 shows the results of an electrothermal model for a single stepof the SCODA cycle in one embodiment. Voltage applied to the fourelectrodes was −120 V, 0 V, 0 V, 0 V. Spreader plate temperature was setto 55° C. (328 K).

FIG. 23 shows SCODA velocity vector plots in one exemplary embodiment ofthe invention.

FIGS. 24A and 24B show predictions of SCODA focusing under theapplication of a DC washing bias in one embodiment. FIG. 24A shows theSCODA velocity field for perfect match target. A circular spot indicatesfinal focus location. FIG. 24B shows the SCODA velocity field for thesingle base mismatch target.

FIG. 25 shows the results of the measurement of temperature dependenceof DNA target mobility through a gel containing immobilizedcomplementary oligonucleotide probes for one exemplary separation.

FIG. 26 shows a time series of affinity SCODA focusing under theapplication of DC bias according to one embodiment. Perfect match DNA istagged with 6-FAM (green) (leading bright line that focuses to a tightspot) and single base mismatch DNA is tagged with Cy5 (red) (trailingbright line that is washed from the gel). Images taken at 3 minuteintervals. The first image was taken immediately following injection.

FIGS. 27A, 27B, 27C, and 27D show the results of performing SCODAfocusing with different concentrations of probes and in the presence orabsence of 200 mM NaCl. Probe concentrations are 100 μM, 10 μM, 1 μM,and 100 μM, respectively. The buffer used in FIGS. 27A, 27B, and 27C was1×TB with 0.2 M NaCl. The buffer used in FIG. 27D was 1×TBE. Differentamounts of target were injected in each of these experiments, and thecamera gain was adjusted to prevent saturation.

FIG. 28 shows an experiment providing an example of phase lag inducedrotations. The field rotation is counterclockwise. It induces aclockwise rotation of the targets in the gel. Images were taken at 5minute intervals.

FIG. 29A shows the focus location under bias for 250 bp and 1000 bpfragments labeled with different fluorescent markers, with squaresindicating data for the application of a 10 V DC bias and circlesindicating data for the application of a 20 V DC bias.

FIG. 29B shows an image of the affinity gel at the end of the run,wherein images showing the location of each fluorescent marker have beensuperimposed.

FIGS. 30A and 30B show respectively the normalized fluorescence signaland the calculated rejection ratio of a 100 nucleotide sequence having asingle base mismatch as compared with a target DNA molecule according toone example.

FIGS. 31A, 31B, and 31C show enrichment of cDNA obtained from an EZH2Y641N mutation from a mixture of wild type and mutant amplicons usingaffinity SCODA with the application of a DC bias. Images were taken at 0minutes (FIG. 31A), 10 minutes (FIG. 31B), and 20 minutes (FIG. 31C).

FIG. 32 shows experimental results for the measurement of mobilityversus temperature for methylated and unmethylated targets. Data pointswere fit to equation (36). Data for the unmethylated target is fit tothe curve on the left; data for the methylated target is fit to thecurve on the right.

FIG. 33 shows the difference between the two mobility versus temperaturecurves which were fit to the data from FIG. 32. The maximum value ofthis difference is at 69.5° C., which is the temperature for maximumseparation while performing affinity SCODA focusing with the applicationof a DC bias.

FIG. 34 shows experimental results for the separation of methylated(6-FAM, green) and unmethylated (Cy5, red) targets by using SCODAfocusing with an applied DC bias.

FIGS. 35A-35D show the separation of differentially methylatedoligonucleotides using affinity SCODA. FIGS. 35A and 35B show theresults of an initial focus before washing unmethylated target from thegel for 10 pmol unmethylated DNA (FIG. 35A) and 0.1 pmol methylated DNA(FIG. 35B). FIGS. 35C and 35D show the results of a second focusingconducted after the unmethylated sequence had been washed from the gelfor unmethylated and methylated target, respectively.

FIGS. 36A-36K show the results of the differential separation of twodifferent sequences in the same affinity matrix using differentoligonucleotide probes. FIG. 36A shows the gel after loading. FIGS. 36Band 36C show focusing at 55° C. after 2 minutes and 4 minutes,respectively. FIGS. 36D and 36E show focusing at 62° C. after 2 minutesand 4 minutes, respectively. FIGS. 36F, 36G, and 36H show focusing ofthe target molecules to an extraction well at the center of the gelafter 0.5 minutes and 1 minute at 55° C. and at 3 minutes after raisingthe temperature to 62° C., respectively.

FIGS. 36I, 36J, and 36K show the application of a washing bias to theright at 55° C. after 6 minutes, 12 minutes and 18 minutes,respectively.

FIG. 37 shows an exemplary plot of mobility versus temperature for amutant nucleic acid, a wild type nucleic acid, and background moleculeswith different binding energies to immobilized probe molecules.

FIGS. 38A and 38B shows an exemplary application of a time-varyingdriving field and a periodically varying, mobility-altering field to amutant nucleic acid, a wild type nucleic acid, and background molecules,in an affinity matrix comprising immobilized probes with a firstaffinity for the wild type nucleic acid which is greater than a secondaffinity for the mutant nucleic acid, which is greater than a thirdaffinity for the background molecules.

FIG. 38C shows an exemplary application of a washing field to a mutantnucleic acid, a wild type nucleic acid, and background molecules, in anaffinity matrix comprising immobilized probes with a first affinity forthe wild type nucleic acid which is greater than a second affinity forthe mutant nucleic acid, which is greater than a third affinity for thebackground molecules

DESCRIPTION

Throughout the following description, specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practiced without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the invention. Accordingly, thespecification and drawings are to be regarded in an illustrative, ratherthan a restrictive, sense.

Methods of the invention provide the ability to isolate low-abundancebiological molecules from a sample. The invention provides for enrichinglow-abundance variants of a biological molecule relative to more common,or wild-type, variants of the molecule. In preferred embodiments,methods of the invention are used to create a subsample in which amolecular species that was present in the original sample inlow-abundance relative to a more common species (e.g., a mutated nucleicacid and its wild-type equivalent) is present in relative high abundancein the subsample.

The skilled artisan will appreciate that there are numerous ways topractice the invention described and claimed herein. However, onepreferred embodiment is exemplified below using a technique calledscodaphoresis or SCODA (Synchronous Coefficient of Drag Alteration).Scodaphoresis refers to methods for moving and/or concentratingparticles in a medium. Scodaphoresis involves exposing particles thatare to be moved and/or concentrated to two time-varying fields orstimuli. A first one of the fields results in a force f(t) that drivesmotion of the particles in the medium. The direction of particle motioncaused by the interaction of the particle with the first field varies intime. The first field may provide a driving force that averages to zeroover an integral number of cycles of the first field.

A second one of the fields alters the mobility of the particles in themedium according to a function g(t). The first and second fields aresuch that f(t) and g(t) have a non-zero correlation over a time periodof interest. Achieving such a non-zero correlation can be achieved invarious ways. In some embodiments, f(t) and g(t) are each time varyingat the same frequency and f(t) and g(t) are synchronized so that thereis a substantially constant phase relationship between f(t) and g(t). Inother embodiments, f(t) has a frequency that is twice that of g(t).

Application of the fields to the particles causes a net drift of theparticles. This net drift can be harnessed to separate particles ofdifferent types or to concentrate (enrich) particles in selected areas,or both. As discussed below, the first and second fields may be of thesame type (homogeneous SCODA) or of different types (heterogeneousSCODA).

As a demonstration of SCODA, consider the case where:ƒ(t)=sin(ωt),g(t)=sin(ωt), and v(ƒ(t),g(t))=ƒ(t)×(μ₀+μ₁ g(t))  (1)where μ₀, is the unperturbed mobility of the particle in the medium andμ₁ is the susceptibility of the mobility to g(t). It can be seen that inthe absence of g(t), the velocity of the particle is given simply byμ₀f(t). Where f(t) is given by Equation (1) there is no net displacementof the particle over a cycle of f(t). Where g(t) is as given above,however, over one cycle, the velocity integrates to yield a distance, d,traveled by the particle of:

$\begin{matrix}{d = {{\int_{t = 0}^{2{\pi/\omega}}{\mu_{1}{\sin^{2}\left( {\omega\; t} \right)}\ {dt}}} = \frac{\mu_{1}\pi}{\omega}}} & (2)\end{matrix}$

Thus, the simultaneous application of the two fields imparts a netmotion to the particle. In this example, the net motion is independentof μ₀.

“Particle” is used herein to mean any microscopic or macroscopic thingthat can be moved by scodaphoresis.

The correlation of f(t) and g(t) may be computed according to a suitablecorrelation function such as:C _(ƒ(t),g(t)=∫) _(T) _(ƒ(t)g(t+λ)dt)  (3)where C is the correlation, T is a period of interest, and λ is aconstant time shift. C must have a non-zero value for some value of λ.

Ideally f(t) and g(t) have a large correlation for efficient operationof SCODA, but some SCODA motion can occur even in cases where the chosenfunctions f(t) and g(t) and the chosen value of λ result in small valuesof C. The velocity of the particle undergoing SCODA motion must be afunction of both f(t) and g(t). Further, the velocity of the particle asa result of the application of f(t) and g(t) together must not be thesame as the sum of the velocities resulting from application of f(t) andg(t) independently. That is:{right arrow over (v)}(ƒ(t),g(t))≠{right arrow over (v)}(ƒ(t),0)+{rightarrow over (v)}(0,g(t+λ))  (4)

One set of conditions which is convenient, but not necessary, forscodaphoresis is:∫_(−∞) ^(∞)ƒ(t)dt=0,∫_(−∞) ^(∞) g(t)dt=0,∫_(−∞) ^(∞) v(ƒ(t),0)dt=0, and ∫_(−∞) ^(∞) v(0,g(t))dt=0  (5)where v(f(t),0) is the velocity of a particle as a function of time whenthe particle is interacting only with the driving field f(t); v(0,g(t))is the velocity of a particle as a function of time when the particle isinteracting only with the mobility-varying field g(t); and,∫_(−∞) ^(∞) v(ƒ(t),g(t))dt≠0  (6)in this case, the two fields, acting independently, do not produce anynet motion of the particle. However, the combined effect of the firstand second fields does result in the particle being moved with a netvelocity.

To optimize SCODA one can select functions f(t) and g(t) so that thefirst order velocity of the particles caused by either f(t) or g(t) iszero (so particles have no net drift), and so that the combination off(t) and g(t) acts on the particles to provide a maximum velocity. Onecan select f(t) and g(t) and a phase shift λ to maximize the integral:∫₀ ^(T) {right arrow over (v)}(ƒ(t),g(t+λ))dt  (7)

The process in this case runs from time 0 to time T or possibly formultiple periods wherein t runs from 0 to T in each period.

It is not necessary that f(t) and g(t) be represented by sinusoidalfunctions, by the same functions, or even by periodic functions. In someembodiments of the invention, f(t) and g(t) are different functions. Insome embodiments of the invention, f(t) and g(t) are not periodic. FIGS.1A through 1H show some examples of functions f(t) and g(t) that couldbe used in specific embodiments of the invention.

FIG. 1A shows a case wherein f(t) and g(t) are both sine functions withf(t) and g(t) in phase. FIG. 1B shows a case where f(t) and g(t) areboth sine functions with f(t) and g(t) out of phase. As described below,the direction in which particles are caused to move can be reversed byaltering the relative phase of f(t) and g(t).

FIG. 1C shows a case where g(t) is unbalanced. In FIG. 1C, f(t) and g(t)are both triangular functions. In FIG. 1C g(t) has a frequency half ofthat of f(t). In FIG. 1D, f(t) has a square waveform while g(t) has asinusoidal waveform. In FIG. 1E, f(t) and g(t) both have substantiallysquare waveforms. In FIG. 1F, f(t) and g(t) have varying frequencies. InFIG. 1G, f(t) is essentially random noise and g(t) has a value of 1 (inarbitrary units) when f(t) exceeds a threshold 7 and has a value of 0otherwise. In FIG. 1H, g(t) has the form of a series of short-durationimpulses.

As another example,

$\begin{matrix}{{{f(t)} = {\sin\left( {\omega\; t} \right)}},{{g(t)} = {{1\mspace{14mu}{for}\mspace{14mu}\frac{2n\;\pi}{\omega}} < t < \frac{\left( {{2n} + 1} \right)\pi}{\omega}}}} & (8)\end{matrix}$where n is any integer or set of integers (e.g. n∈{1, 2, 3, . . . } orn∈{2, 4, 6, . . . } or n∈{1, 4, 7, . . . }. The integers n do not needto be regularly spaced apart. For example, the methods of the inventioncould be made to work in a case wherein the set of integers n consistsof a non-periodic series. An otherwise periodic waveform f(t) or g(t)could be made aperiodic by randomly omitting troughs (or peaks) of thewaveform, for example.

FIG. 1I illustrates a case where f(t) has a frequency twice that ofg(t). The waveforms of FIG. 1I can produce SCODA motion, for example,where the mobility of particles varies in response to |g(t)|. It can beseen that |g(t)| has larger values for positive-going peaks of f(t) thanfor negative-going peaks of f(t).

While the waveforms shown in most of FIGS. 1A to 1I are symmetrical(i.e. they have the same overall form if inverted in spatial direction)this is not mandatory. f(t) could, in general, be asymmetrical.

Driving Fields

f(t) is referred to herein as a driving function because it drivesmotion of the particles in the medium. In different embodiments of theinvention, f(t) is produced by fields of different types. For example,f(t) may be produced by any of:

a time-varying electric field;

a time-varying magnetic field;

a time-varying flow in the medium;

a time-varying density gradient of some species in the medium;

a time-varying gravitational or acceleration field (which may beobtained, for example by accelerating a medium containing particles andperiodically changing an orientation of the medium relative to thedirection of the gravitational or acceleration field);

or the like.

In some embodiments, f(t) applies a force to particles that alternatesin direction wherein the magnitude of the force is the same in eachdirection. In other embodiments, f(t) combines a component thatalternates in direction and a bias component that does not alternate indirection such that the magnitude of the force applied to particles islarger in one direction than in the other. The bias component may betermed a DC component while the alternating component may be termed anAC component.

The driving field is selected to interact with the particles ofinterest. For example:

Where the particles are electrically charged particles (ions forexample), an electric field may be used for the driving field.Electrically neutral particles may be made responsive to an electricfield by binding charged particles to the electrically neutralparticles. In some cases an electrically neutral particle, such as aneutral molecule, can be carried by a charged particle, such as acharged molecule. For example, neutral proteins that interact withcharged micelles may be driven by an electrical driving field throughthe interaction with the driving field and the micelles.

Where the particles have dielectric constants different from that of themedium, an electric field having a time-varying gradient can drivemotion of the particles through the medium by dielectrophoresis.

Where the particles contain magnetic material (for example, whereparticles of interest can be caused to bind to small beads of a typeaffected by magnetic forces, for example ferromagnetic beads) a magneticfield may be used for the driving field.

Where the particles have magnetic susceptibilities different from thatof the medium then a gradient in a magnetic field may be used to drivemotion of the particles relative to the medium by magnetophoresis.

Where the particles have densities different from that of the mediumthen a gravitational or other acceleration acting on the particles maydrive motion of the particles relative to the medium. An AC accelerationis provided in some embodiments by exposing the medium to an acousticfield.

The driving field may directly apply a force to the particles or mayindirectly cause motion of the particles. As an example of the latter,the driving field may cause living particles (mobile bacteria forexample) to move in response to their own preference for certainenvironments. For example, some organisms will swim toward light,chemical gradients, or magnetic fields (these phenomena are known aschemotaxis, phototaxis, and magnetotaxis respectively).

Mobility-Varying Fields

The mobility of particles may by altered according to any of a widevariety of mechanisms. For example:

changing a temperature of the medium;

exposing the particles to light or other radiation having an intensityand/or polarization and/or wavelength that varies in time with thedriving field;

applying an electric field to the portion of the medium through whichthe particles are passing;

applying a magnetic field to the medium through which the particles arepassing (the magnetic field may, for example, alter an orientation of amagnetic dipole associated with the particle and thereby affect acoefficient of drag of the particle or alter a viscosity of the mediumwhich may comprise a suitable magneto-rheological fluid);

applying an acoustic signal to the portion of the medium through whichthe particles are passing;

causing a cyclic change in concentration of a species in the medium;

exploiting electroosmotic effects;

causing cyclic chemical changes in the medium;

causing the particles to cyclically bind and unbind to other particlesin or components of the medium;

varying a hydrostatic pressure experienced by the medium;

varying physical dimensions of the medium to cause a change in aneffective drag experienced by particles in the medium;

applying magnetic fields to the medium.

Any effect that varies the mobility of a particle in response to adriving field, such as an electrophoretic driving field, can be used.

In some embodiments of the invention, the mobility of particles isvaried by exploiting non-linearities in the relationship between thevelocity of a particle and the intensity of the driving field. Someembodiments apply a second driving field having a component actingperpendicular to the direction of the first driving field but afrequency half that of the first driving field. Applied by itself, sucha second driving field would simply cause particles to oscillate backand forth in a direction perpendicular to the direction of the maindriving field. When applied together with the main driving field,however, such a second driving field can cause particles to have higheraverage speeds relative to the medium for one direction of the maindriving field than for the other direction of the main driving field.This results in a net drift of the particles because of the non-linearrelationship between particle mobility and particle speed. In someembodiments the main driving field has a symmetrical waveform, such as asinusoidal, triangular or square waveform.

A temperature of the medium in which the particles are situated may bealtered in time with the driving field. The changing temperature mayresult in a change in one or more of a conformation of the particles, aviscosity of the medium, a strength of interaction between the particlesand the medium, some combination of these, or the like. The result isthat the mobility of the particles is altered by the change intemperature. The temperature of regions in a medium may be controlled inany suitable manner including:

directing radiation at the portion of the medium to heat that portion ofthe medium;

energizing heaters or coolers in thermal contact with the portion of themedium;

causing endothermic or exothermic chemical reactions to occur in theportion of the medium (or in a location that is in thermal contact withthe portion of the medium); and,

the like.

In some embodiments of the invention the medium comprises a materialthat absorbs radiation and releases the absorbed radiation energy asheat. In some embodiment, localized heating of the medium in thevicinity of the particles being moved is achieved by irradiating theparticles with electromagnetic radiation having a wavelength that isabsorbed by the particles themselves and released as heat. In suchembodiments it can be advantageous to select a wavelength for theradiation that is not absorbed or converted to heat significantly byconstituents of the medium so that heating is local to the particles.

Some examples of particles that have mobilities that vary withtemperature are: proteins that can be cyclically denatured or caused tofold in different ways by cyclically changing the temperature; and DNAthat can be cyclically denatured.

Exposing the area of the medium in which the particles are travelling toradiation changes one or more of: a conformation of the particles, aviscosity of the medium, a strength of interaction between the particlesand the medium, some combination of these, or the like. The result isthat the mobility of the particles is altered by changes in theintensity and/or polarization and/or wavelength of the appliedradiation. Some examples of particles that have mobilities that can becaused to change by applying light are molecules such as azobenzene orspiro-pyrans, that can be caused to undergo reversible changes inconformation by applying light. Another example of the use of light tovary the mobilities of particles in a medium is the application of lightto cause partial cross-linking of polymers in a medium containingpolymers.

The intensity of an electric field applied to the medium may be variedin time with the driving field. In some media the mobility of particlesof certain types varies with the applied electric field. In some mediathe particle velocity varies non-linearly with the applied electricfield.

The mobility of particles in a medium may vary with the intensity of anacoustic field applied to the medium. In some cases, an acousticstanding waves in a solution or other medium may cause transientdifferences in local properties of the medium (e.g. electricalresistivity) experienced by particles in the medium thus leading tolocal inhomogeneity in the driving field (e.g. a driving electricfield).

Where mobility of particles is controlled by altering a concentration ofa species, the species having the varying concentration may, forexample, be a species that binds to the particles or a species thataffects binding of the particles to some other species or to a surfaceor other adjacent structure. The species may directly affect a viscosityof the medium.

As an example of the use of electroosmotic effects to control particlemobility, consider the case where the medium in which the particles aremoving is a solution containing one or more polymers. In such solutions,an applied electric field can cause bulk fluid flow. Such a flow couldbe controlled to provide a perturbing stimulus to a pressure or flowinduced driving force, or as a perturbation to an electrical drivingforce, possibly exploiting non-linearities in the onset ofelectroosmotic flow.

Chemical changes that are exploited to control particle mobility may,for example, induce changes in one or more of:

a conformation of the particles;

a conformation of some other species;

binding of the particles to one another or to other species orstructures in the medium;

binding of species in the medium to one another;

viscosity of the medium; or

the like.

The chemical changes may be induced optically, for example, by opticallyinducing cross-linking or by optically inducing oxidation or reductionof photoactive molecules such as ferrocene. The chemical changes may beinduced by introducing chemical species into the medium. The chemicalchanges may include one or more of changes: that alter the pH of themedium; changes that result in changes in the concentration of one ormore chemical species in the medium; or the like.

Particle mobility may be affected by applied magnetic fields accordingto any of a variety of mechanisms. For example:

The medium may contain small magnetic beads. The beads may be linked topolymers in a polymer matrix. By applying a magnetic field, the beadsmay be pulled away from a path of the particles, thereby reducing aneffective viscosity of the medium experienced by the particles.

The medium could be a magneto-rheological fluid having a viscosity thatvaries with applied magnetic field.

A magnetic field may be used to cause medium viscosity to vary accordingto a two-dimensional pattern. The magnetic field could change in time insuch a manner that the viscosity of the medium varies with position andvaries in time in a manner that provides a synchronous perturbation to aperiodic driving force. As another example, where the particlesthemselves are magnetic, transport and concentration of the particlescould be affected by a magnetic field. The particles could be drivenelectrophoretically. The magnetic field could be switched onperiodically to drive the particles toward a drag-inducing surface, orrelease them from such a surface. The magnetic field could also be usedto make the particles aggregate.

Particles

The methods of the invention may be applied to particles of virtuallyany kind including molecules, ions, and larger particulates. Somenon-limiting examples of particles which may be moved, concentratedand/or extracted through use of the methods of the invention are:

electrically charged or neutral biomacromolecules such as proteins,nucleic acids (RNA, DNA), and suitable lipids; long polymers;polypeptides;

aggregations of molecules such as micelles or other supramolecularassemblies;

any particles to which magnetic beads or electrically-charged beads canbe attached;

living microorganisms; and,

the like.

In particular the invention is effective at separating nucleic acids,which may be single-stranded or double stranded, and may vary in lengthfrom thousands of bases, to hundreds of bases, to tens of bases. In oneinstance, the invention is used to separate or enrich so called shortnucleic acids, having 500 or fewer, e.g., 200 or fewer, e.g., 100 orfewer, e.g., 50 or fewer bases. Short nucleic acids are commonly theresult of cellular breakdown, and may be found, for example, incell-free samples (e.g., blood plasma, urine), formalin-fixed samples,or forensic samples.

For any particular type of particle, one can attempt to identify asuitable driving field, medium, and mobility-altering field. Since manybiomacromolecules can be electrically charged, it is often suitable touse a time-varying electrical field as the driving field when applyingthe invention to moving and/or concentrating such particles. Further,there are well developed techniques for causing magnetic beads to bondto specific biological materials. Where it is desired to move and/orconcentrate materials which can be caused to bond to magnetic beads thenmagnetic fields may be used as driving fields.

Media

The medium is selected to be a medium through which the particles canmove and also a medium wherein the mobility of the particles can bealtered by applying a suitable mobility-altering field. The medium maycomprise, for example:

a gel, such as an agarose gel or a performance optimized polymer (POP)gel (available from Perkin Elmer Corporation);

a solution, aqueous or otherwise;

entangled liquid solutions of polymers;

viscous or dense solutions;

solutions of polymers designed to bind specifically to the molecules (orother particles) whose motion is to be directed;

acrylamide, linear poly-acrylamide;

micro-fabricated structures such as arrays of posts and the like, withspacing such that the particles of interest can be entangled or retardedby frequent collision or interaction with the micro-fabricatedstructure;

structures designed to interact with molecules by means of entropictrapping (see, e.g. Craighead et al., in Science 12 May 2000 Vol. 288);

high viscosity fluids such as PLURONIC™ F127 (available from BASF);

water; or

the like.

The medium is chosen to have characteristics suitable for the particlesbeing moved. Where the particles are particles of DNA then suitablepolymer gels are the media currently preferred by the inventors. In somespecific embodiments of the invention the particles comprise DNA and themedium comprises an agarose gel or a suitable aqueous solution. In someembodiments the aqueous solution is a bacterial growth medium mixed witha gel such as an agarose gel.

2D Scodaphoresis

In some embodiments, the particles are constrained to move on atwo-dimensional (2D) surface. In some embodiments the 2D surface isplanar. The 2D surface is not necessarily planar. In some embodiments,the 2D surface comprises a relatively thin layer of a medium, such as agel. In some embodiments the medium is free-standing. The medium may besupported on a substrate. The substrate may comprise a sheet of glass ora suitable plastic such as mylar, for example. In some embodiments the2D layer of medium is sandwiched between the surfaces of two substrates.Where the medium has an exposed surface, the surface may be in air oranother gaseous atmosphere or submerged in a liquid such as a suitablebuffer, an oil, or the like. In some currently preferred embodiments,the medium comprises a layer of a gel sandwiched between two layers ofthicker gel. In an example embodiment, particles move in a layer of a 1%w/v agarose gel sandwiched between two layers of 3% w/v agarose gel.

In some embodiments of the invention, a 2D surface in which particlestravel may be provided by a layer within a medium which has anon-uniform viscosity or a non-uniform concentration of a species thatreduces (or increases) a mobility of the particles. The viscosity orconcentration gradient cause particles to remain in the relatively thinlayer within the medium or on a surface of the medium.

3D Scodaphoresis

SCODA may be used to concentrate particles in three dimensions. This maybe achieved in various ways. In some embodiments, 2D SCODA is performedin a plane. The 2D SCODA may be performed using the electrophoreticSCODA method described below, for example, Z electrodes placed above andbelow the plane could apply an electric field that tends to drive anyparticles that begin to move out of the plane back into the plane.

3D SCODA could also be performed by providing a 6 electrode arrangement,where each electrode is placed on the surface of a body of a medium suchas a gel. Defining X Y and Z axes of such a cube, 2D SCODA would then berun on the 4 electrodes in the XY plane, then the 4 electrodes in the YZplane, then the 4 electrodes in the XZ plane, then repeating in the XYplane and so forth. This would produce a net 3D focusing effect, with anet SCODA force that is radial in three dimensions, but about % asstrong as the 2D SCODA force for the same electrode voltages.

Samples

A variety of fluidic samples can be enriched using methods of theinvention. Additionally, solid samples may be solubilized or suspendedand then enriched. Suitable biological samples may include, but are notlimited to, cultures, blood, plasma, serum, saliva, cerebral spinalfluid, pleural fluid, milk, lymph, sputum, semen, urine, stool, tears,saliva, sweat, needle aspirates, external sections of the skin,respiratory, intestinal, and genitourinary tracts, tumors, organs, cellcultures or cell culture constituents, or tissue sections. In someembodiments, the biological sample may be analyzed as is, that is,without additional preparation. In an alternate embodiment, harvestand/or isolation of materials of interest may be performed prior toanalysis.

A sample may include any of the aforementioned samples regardless oftheir physical condition, such as, but not limited to, being frozen orstained or otherwise treated. In some embodiments, a biological samplemay include compounds which are not naturally intermixed with the samplesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics, or the like.

In some embodiments, a biological sample may include a tissue sample, awhole cell, a cell constituent, a cytospin, or a cell smear. A tissuesample may include a collection of similar cells obtained from a tissueof a biological subject that may have a similar function. In someembodiments, a tissue sample may include a collection of similar cellsobtained from a tissue of a human. Suitable examples of human tissuesinclude, but are not limited to, (1) epithelium; (2) the connectivetissues, including blood vessels, bone and cartilage; (3) muscle tissue;and (4) nerve tissue. The source of the tissue sample may be solidtissue obtained from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,or interstitial fluid; or cells from any time in gestation ordevelopment of the subject. In some embodiments, the tissue sample mayinclude primary or cultured cells or cell lines. In some embodiments, abiological sample includes tissue sections from healthy or diseasedtissue samples (e.g., tissue section from colon, breast tissue,prostate, lung, etc.). A tissue section may include a single part orpiece of a tissue sample, for example, a thin slice of tissue or cellscut from a tissue sample.

In some embodiments, a biological sample may be recovered from a solidsupport and suspended or solubilized prior to being used with methods ofthe invention. A solid support may include microarrays (e.g., DNA or RNAmicroarrays), gels, blots, glass slides, beads, swabs or ELISA plates.In some embodiments, a biological sample may be adhered to a membraneselected from nylon, nitrocellulose, and polyvinylidene difluoride. Insome embodiments, the solid support may include a plastic surfaceselected from polystyrene, polycarbonate, and polypropylene. In someembodiments the biological sample is recovered from a formalin-fixedsample, e.g., a formalin-fixed paraffin-embedded (FFPE) sample.

A biological sample may be of prokaryotic origin or eukaryotic origin(e.g., insects, protozoa, birds, fish, reptiles). In some embodiments,the biological sample is mammalian (e.g., rat, mouse, cow, horse, pig,dog, cat, donkey, guinea pig, or rabbit). In certain embodiments, thebiological sample is of primate origin (e.g., example, chimpanzee, orhuman). The samples may be forensic samples including, but not limitedto, blood samples, saliva samples, urine samples, feces samples,microbial samples, pathogen samples, forensic biological samples, crimescene biological samples, drug/alcohol samples, chemicals (e.g.,explosives), and residues.

Additional Analysis of Particles

In some instances, enriched samples produced with the methods andapparatus of the invention will be additionally analyzed or processed.For example, the resultant enriched sample may be amplified, hybridized,stored, lyophilized, or sequenced.

Where the enriched sample contains nucleic acids, the sample may beamplified using Polymerase Chain Reaction (PCR) technologies. A typicalPCR reaction includes multiple amplification steps, or cycles thatselectively amplify a targeted nucleic acid species. Additionalreferences describe the PCR process, and common variations thereof, suchas quantitative PCR (QPCR), real-time QPCR, reverse transcription PCR(RT-PCR) and quantitative reverse transcription PCR (QRT-PCR). PCRinstruments and reagents are commercially available from suppliers suchas Roche Molecular Diagnostics (Pleasanton, Calif.).

A typical PCR reaction includes three steps: a denaturing step in whicha targeted nucleic acid is denatured; an annealing step in which a setof PCR primers (forward and backward primers) anneal to complementaryDNA strands; and an elongation step in which a thermostable DNApolymerase elongates the primers. By repeating this step multiple times,a DNA fragment is amplified to produce an amplicon, corresponding to thetargeted DNA sequence. Typical PCR reactions include 30 or more cyclesof denaturation, annealing and elongation. In many cases, the annealingand elongation steps can be performed concurrently, in which case thecycle contains only two steps. Using PCR amplification, it is possibleto amplify the targeted nucleic acid exponentially.

However, as discussed in the background of this application, PCRamplification introduces errors into the amplified nucleic acidproducts. In some instances, the error rate is of the same magnitude asthe incidence of target nucleic acids in the sample. In these instancesif PCR amplification is used, it is done after enrichment to avoidcreating erroneous target nucleic acids. In some embodiments, where thePCR error rate is acceptable compared to the incidence of target nucleicacids in the sample, it is beneficial to do some PCR on the sample priorto enrichment, to boost the total number of target nucleic acids in thesample. In practice, PCR prior to enrichment is limited to fewer than 20cycles, e.g., 15 or fewer cycles, e.g., 10 or fewer cycles, e.g., 5 orfewer cycles, in order to limit the introduction of errors. Afterenrichment, the enriched target nucleic acids may be amplified forfurther processing with 20 or more, e.g., 25 or more, e.g., 30 or more,e.g., 40 or more PCR cycles.

Several methods are available to identify target nucleic acids (e.g.,variant nucleic acids, e.g., mutations) that have been enriched usingmethods and apparatus of the invention. In some instances an enrichedsample may be analyzed with a hybridization probe. Typically, a labeledsingle stranded polynucleotide, which is complementary to all or part ofthe targeted sequence, is exposed to the sample, a wash step isperformed, and then the sample is observed for the presence of thelabel. In some instances, amplification and hybrid probe analysis may beperformed simultaneously, e.g., using quantitative PCR.

In other instances the complimentary polynucleotide probes may beimmobilized on a solid support. In this instance, hybrid probe analysistypically includes (1) labeling nucleic acids in the enriched sample,(2) pre-hybridization treatment to increase accessibility ofsupport-bound probes and to reduce nonspecific binding; (3)hybridization of the labeled nucleic acids to the surface-boundpolynucleotides, typically under high stringency conditions; (4)post-hybridization washes to remove nucleic acid fragments not bound tothe solid support polynucleotides; and (5) detection of the hybridized,labeled nucleic acids. Detection may be done, for example byfluorescence detection, however other methods may be used, dependingupon the nature of the label.

In some embodiments, an enriched sample containing multiple targetnucleic acids may be identified with a multiplex protocol designed toidentify multiple specific mutations of interest. For example, singlenucleotide polymorphisms (SNPs) among the target nucleic acids may bedetermined with a single base extension kit, such as SNAPSHOT™ availablefrom Applied Biosystems (Life Technologies, Carlsbad, Calif.). Usingthis kit, the enriched sample will be mixed with a set of primers ofvarying length and sequence, each primer being complimentary todifferent loci on the target nucleic acids. Upon mixing, the primerswill hybridize with a specific target nucleic acid, forming a duplexwith a 3′ terminus adjacent to the SNP. In the presence of a polymerase,a single fluorescently-labeled base is added to the duplex and theresulting populations of fluorescently-labeled moieties can becharacterized by length and label color (e.g., using Sanger sequencings,for example GENESCAN™ analysis, Applied Biosystems) to determine thepresence and amount of the mutations.

Another method that can be used to identify nucleic acids in theenriched sample is genetic sequencing. Sequencing may be by any methodknown in the art. DNA sequencing techniques include classic dideoxysequencing reactions (Sanger method) using labeled terminators orprimers and gel separation in slab or capillary, sequencing by synthesisusing reversibly terminated labeled nucleotides, pyrosequencing, 454™sequencing, allele specific hybridization to a library of labeledoligonucleotide probes, sequencing by synthesis using allele specifichybridization to a library of labeled clones that is followed byligation, real time monitoring of the incorporation of labelednucleotides during a polymerization step, polony sequencing, and SOLiD™sequencing.

In preferred embodiments, nucleic acids enriched with methods of theinvention may be sequenced using next-generation sequencing. Forexample, 454™ sequencing, available from Roche (Branford, Conn.), may beused to quickly and accurately sequence enriched nucleic acid samples.(See Margulies, M et al. 2005, Nature, 437, 376-380, incorporated hereinby reference in its entirety.) 454™ sequencing involves two steps. Inthe first step, DNA is sheared into fragments of approximately 300-800base pairs, and the fragments are blunt ended. Oligonucleotide adaptorsare then ligated to the ends of the fragments. The adaptors serve asprimers for amplification and sequencing of the fragments. The fragmentsare then attached to DNA capture beads, e.g., streptavidin-coated beadsusing, e.g., Adaptor B, which contains 5′-biotin tag. The fragmentsattached to the beads are PCR amplified within droplets of an oil-wateremulsion to make multiple copies of DNA fragments on each bead. In thesecond step, the beads are captured in picoliter wells. Finally,pyrosequencing is performed on each DNA fragment in parallel. Asnucleotides are added, a light signal is generated and recorded by a CCDcamera in the instrument. The signal strength is proportional to thenumber of nucleotides incorporated. The signals are then analyzed andcorrelated to determine the sequence.

Alternatively, ION TORRENT™ sequencing systems, available from LifeTechnologies (Carlsbad, Calif.) may be used to directly obtain thesequences of the enriched nucleic acids. Among other references, themethods and devices of ION TORRENT™ sequencing are disclosed in U.S.patent application numbers 2009/0026082, 2009/0127589, 2010/0035252,2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559,2010/0300895, 2010/0301398, and 2010/0304982, the content of each ofwhich is incorporated by reference herein in its entirety. In IONTORRENT™ sequencing, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments are then attached to a surface at a concentration such thatthe fragments are individually resolvable. Addition of one or morenucleotides releases a proton (H⁺), which is detected and recorded in asequencing instrument. The signal strength is proportional to the numberof nucleotides incorporated. The signals are then analyzed andcorrelated to determine the sequence.

Another example of a next-generation sequencing technology that can beused to sequence enriched nucleic acids is ILLUMINA™ sequencing,available from Illumina, Inc, (San Diego, Calif.). ILLUMINA™ sequencingamplifies DNA on a solid surface using fold-back PCR and anchoredprimers. The DNA is then fragmented, and adapters are added to the 5′and 3′ ends of the fragments. Next, fragments are attached to thesurface of flow cell channels, and the DNA is extended and bridgeamplified. This process results in several million clusters ofapproximately 1,000 copies of single-stranded DNA molecules of the sametemplate in each channel of the flow cell. Using primers, DNApolymerase, and four fluorophore-labeled, reversibly-terminatingnucleotides, the copies are then sequentially sequenced andfluorescence-imaged to determine the added nucleotide. The 3′terminators and fluorophores from each incorporated base aresubsequently removed, and the incorporation, detection andidentification steps are repeated to read out the next nucleotide.

In some instances, the enriched nucleic acids will be identified usingmass spectrometry. Mass spectrometry uses a combination of electricand/or magnetic fields to cause nucleic acid ions (or pieces of) tofollow specific trajectories (or to have specific flight times)depending on their individual mass (m) and charge (z). In addition, byarranging collisions of a parent molecular ion with other particles(e.g. argon atoms), the molecular ion may be fragmented formingsecondary ions by the so-called collision induced dissociation (CID).The fragmentation pattern/pathway very often allows the derivation ofdetailed structural information. The structural information may be usedto determine the sequence of the nucleic acid. Nucleic acids aredifficult to volatilize, however. Using techniques such aselectrospray/ionspray (ES) and matrix-assisted laserdesorption/ionization (MALDI), nucleic acids can be volatilized,ionized, and characterized by their mass-to-charge profile.Additionally, DNA massarrays, such as offered by Sequenom (San Diego,Calif.), can be used to facilitate MALDI mass spectrometric analysis bytagging complimentary nucleic acids with easily-detected mass labels.

Control Systems

Any suitable control mechanism may be used to cause a driving field anda mobility-varying field to be applied in a coordinated manner to causeparticles to move by SCODA. In some embodiments of the invention, thetime-variation of the driving field and the mobility-varying field arederived directly from a common source such that their effects on theparticles are correlated. In other embodiments of the invention thedriving and mobility-varying fields are generated under the control of acontroller such as a hard-wired controller, a programmable controller, ageneral purpose computer equipped with suitable interface electronics orthe like. Any suitable control mechanism including those known to thoseskilled in the art of designing scientific equipment may be applied.

EXAMPLES

The following examples illustrate various specific embodiments of theinvention. These embodiments of the invention are considered to beindividually inventive. Some of these examples summarize experimentsthat have been performed and others are prophetic examples.

Example 1: Electrophoretic Concentration of Particles by SCODA

Consider an electrically charged particle that has an electrophoreticmobility, μ in an electric field given by {right arrow over(E)}=cos(ωt)EÊ where Ê is a unit vector. By definition, the particlewill move with a velocity given by:{right arrow over (v)}=μ cos(ωt)E ₀ {right arrow over (E)}  (9)

From Equation (9), {right arrow over (v)} has a time average of zero. Ifμ varies as a function of time and the Fourier transform of μ has acomponent proportional to cos(ωt) then the time average of v(t) may notbe zero. As a simple example, consider the case where:μ(t)=μ₀+μ₁ cos(ωt)  (10)

In this case, the time average of v(t) is:{right arrow over (v)}=½μ₁ E ₀ Ê  (11)

This demonstrates the basic principle that there can be a non-zeroelectrophoretic drift even if the time average of the applied electricfield is zero.

Now consider the case where the mobility of a particle is a function ofelectric field strength. While virtually any nonlinearity can beemployed, consider the case where a particle's velocity is parallel tothe direction of a driving electric field and the particle's speed isgiven by:v=kE ²  (12)where k is a constant and E is the magnitude of the electric field. Inthis case, the particle's speed is proportional to the square of themagnitude of the electric field. The effective mobility of the particle(i.e. the relationship between small changes in drift velocity, d{rightarrow over (v)}, and small changes in the electric field, d{right arrowover (E)}) varies with the magnitude of the applied electric field.

In Cartesian coordinates:

$\begin{matrix}{{{dv}_{x} = {{\frac{\partial v_{x}}{\partial E_{x}}{dE}_{x}} + {\frac{\partial v_{x}}{\partial E_{y}}{dE}_{y}}}}{and}{{dv}_{y} = {{\frac{\partial v_{y}}{\partial E_{x}}{dE}_{x}} + {\frac{\partial v_{y}}{\partial E_{y}}{dE}_{y}}}}} & (13)\end{matrix}$

Where the particle speed varies with the electric field as in Equation(12), Equation (13) reduces to:

$\begin{matrix}{{{dv}_{x} = {k\left\lbrack {{\left( {E + \frac{E_{x}^{2}}{E}} \right){dE}_{x}} + {\left( \frac{E_{x}E_{y}}{E} \right){dE}_{y}}} \right\rbrack}},{and}} & (14) \\{{dv}_{y} = {k\left\lbrack {{\left( \frac{E_{x}E_{y}}{E} \right){dE}_{x}} + {\left( {E + \frac{E_{y}^{2}}{E}} \right){dE}_{y}}} \right\rbrack}} & (15)\end{matrix}$

To help interpret this, consider the case where E_(y)=0 such thatE_(x)=E. In this case Equations (14) and (15) become:dv _(x)=2kEdE _(x) and dv _(y) =kEdE _(y)  (16)

From Equation (16) one can see that the influence on the particlevelocity of perturbations of the electric field has a magnitudeproportional to that of the ambient field. A perturbation having thesame direction as the electric field has twice the influence on theparticle velocity as a perturbation perpendicular to the electric field.

This can be exploited to provide an applied electric field that causesparticles to be concentrated. Consider a plane wherein an appliedelectric field has a constant magnitude, E, and the electric fieldrotates in direction at an angular frequency ω so that the components ofthe electric field in x and y directions are given by:E _(x) =E cos(ωt) and E _(y)=sin(ωt)  (17)

Substituting the values from Equation (17) into Equations (14) and (15)yields a result which is the sum of constant terms, sine and cosineterms having an angular frequency ω, and sine and cosine terms having anangular frequency 2ω. A frame of reference can be selected such thatonly the cosine terms having an angular frequency of 2ω contribute tonet particle drift. Evaluating only these terms yields:

$\begin{matrix}{{{dv}_{x} = {{\frac{kE}{2}\left\lbrack {\cos\left( {2\omega\; t} \right)} \right\rbrack}{dE}_{x}}},{{dv}_{y} = {{\frac{kE}{2}\left\lbrack {\cos\left( {2\omega\; t} \right)} \right\rbrack}{dE}_{y}}}} & (18)\end{matrix}$

If a perturbing electric field having the form of a quadrupole fieldthat varies with a frequency 2ω is added to the basic electric fieldspecified by Equation (17) then a net drift of particles can be caused.For a perturbing electric field given by:dE _(x) =−dE _(q) x cos(2ωt) and dE _(y) =dE _(q) y cos(2ωt)  (19)

it can be shown that:

$\begin{matrix}{\overset{\_}{d\overset{\rightarrow}{v}} = {\frac{{kEdE}_{q}}{4}\overset{\rightarrow}{r}}} & (20)\end{matrix}$

Equation (20) shows that for charged particles at all positions {rightarrow over (r)} there is a time-averaged drift toward the origin with aspeed proportional to k, the coefficient that specifies thefield-dependence of the mobility, E, the strength of the rotating field,and dEq, the strength of the perturbing quadrupole field.

The above calculation is for a case where the perturbing quadrupolefield has a magnitude that is small in comparison to the rotating field.This is not necessary in general. FIG. 2 shows the result of a numericalsimulation of the path of a particle in a case where the rotatingelectric field and quadrupole electric field are similar in magnitude.Motion begins at the top right hand side of FIG. 2 and progresses towardthe bottom left over a period of 200 seconds. The applied electricfields are as described in Table I below. Each loop in the spiral pathcorresponds to a cycle of 12 voltage patterns each applied for 1 second.The uniform field amplitude is 3845 V/m at the origin (center of theelectrode pattern). At the same location, the magnitude of thequadrupole component of the electric field is 4.2×105 V/m² or about 4200V/m at a location 1 mm from the origin.

In many situations it is advantageous to concentrate particles inregions that are free of electrodes. Electrochemical processes atelectrodes can cause damage to DNA and other sensitive materials. Anelectrical field that provides a particle focusing effect, as describedabove, can be provided without the need for electrodes at the locationin which the particles become concentrated.

One can estimate the size of the spot into which particles can beconcentrated from the Einstein-Smoluchowsky equation for diffusion withdrift. A characteristic length scale, R, for the radius of aconcentrated spot is given by:

$\begin{matrix}{R \propto \sqrt{\frac{D}{\mu_{s}}}} & (21)\end{matrix}$where D is the diffusion coefficient for the particles and μ_(s) isgiven by kEE_(q)/4.

FIG. 3A shows apparatus 10 having a simple arrangement that can be usedto practice the invention. A layer 11 of a medium, which may be a gel,such as an agarose gel, is located between four symmetrically arrangedelectrodes 12A, 12B, 12C, and 12D (collectively electrodes 12). It hasbeen found to be desirable to provide electrodes 12 in the form of meshelectrodes. A power supply 14 applies individually controllableelectrical potentials V1, V2, V3, and V4 to electrodes 12A through 12Drespectively. Since it is the relative potentials of electrodes 12Athrough 12D that is significant, any one of electrodes 12A to 12D may beheld at a convenient fixed voltage, such as 0 volts, while the voltagesapplied to the other electrodes are varied, if desired.

It is generally desirable to control the potentials applied to theelectrodes to help stabilize the electric stimuli against smallfluctuations due to changing temperature or changing power supplycharacteristics. Separate electrical potential sensing electrodes may beincorporated to provide feedback to a controller representing the actualelectrical potential being applied. FIG. 3B is a schematic view of anapparatus comprising mesh electrodes 12A, 12B, 12C, and 12D and separatepotential sensing electrodes 13A, 13B, 13C, and 13D (collectivelyelectrodes 13). Large buffer reservoirs 15 maintain an ample supply ofbuffer against evaporation for long runs. Insulating barriers 16separate adjacent reservoirs 15 electrically. Electrodes 13 are locatedin buffer reservoirs 15 and monitor the potential in the buffer.Feedback from electrodes 13 allows a suitably configured controller 14to automatically adjust the voltages on mesh electrodes 12 to compensatefor varying voltage drops across the mesh electrodes/buffer interface.

The magnitude of the applied voltage is chosen to match the size of theapparatus and the particles being separated. For DNA separations inagarose gels electric driving fields of approximately 50V/cm have beenfound to give satisfactory performance. The current supplied will dependupon the electrical conductivity and dimensions of the medium.

The application of the potentials causes electrically charged particlesin medium 11 to move toward a central region 18. FIG. 3A shows groups17A and 17B of particles moving toward concentration region 18. As notedabove, the precise waveform according to which the applied electricfields vary is not critical to the operation of the invention. In aprototype embodiment of the invention, the potential variation ofEquations (16) and (18) was approximated by a series of patterns ofdiscrete voltages applied to electrodes 12A through 12D. In theprototype, each cycle was made up of 12 patterns that were each appliedfor 1 second before moving to the next pattern. Table 1 shows thevoltages applied for each pattern.

TABLE 1 Applied voltages for scadophoresis apparatus of FIG. 3A. VoltagePatterns Pat- Electrode 12A Electrode 12B Electrode 12C Electrode 12Dtern (V) (V) (V) (V) 1 0 −66 0 −198 2 132 132 0 0 3 132 198 0 198 4 132198 0 198 5 132 0 0 132 6 0 −198 0 −66 7 0 −198 0 −66 8 −132 −132 0 0 9−132 66 0 66 10 −132 66 0 66 11 −132 0 0 −132 12 0 −66 0 −198

In the prototype embodiment of the invention illustrated schematicallyin FIG. 3C, medium 11 was in the form of a gel slab made up of 8-11 mlof 0.25% agarose gel (Agarose 2125 OmniPur available from EMD Chemicalsof Gibstown N.J., USA) forming a 3.8 cm square on an acrylic base in a0.1× Tris-acetate-EDTA buffer. Four electrodes were submerged in thegel. Each electrode extended across one third of one side of the gelboat approximately 2.5 mm up from the bottom of the gel boat. DNA wasprepared by mixing 8 μl of 500 μg/ml λ phage DNA (48,502 bp, part No.N3011L available from New England Biolabs of Beverly Mass., USA) with 12μl 0.1×TAE. 5 μl spots of the DNA were pipetted directly onto the gelafter the gel had set. A thin covering of TAE was placed on the gel. Thevoltage patterns of Table 1 were applied to the electrodes. It was foundthat the DNA spots were all carried to a central area of the gel.

FIG. 4A is an example plot of measured DNA velocity as a function ofapplied electric field for the λ DNA used in the prototype embodiment.FIG. 4B is a plot showing time averaged drift velocity (averaged over 15minutes) of the DNA as a function of the radial distance from the originto which the DNA converged. FIG. 4B includes curve b which is anumerical estimate of the trajectory of a particle starting at alocation on the X-axis and curve c which is a numerical estimate of thetrajectory of a particle starting at a location X=Y=1.5 cm from theorigin.

FIG. 4C is a plot showing the measured DNA spot distance from the originas a function of time compared to numerical and analytical predictions.The spot position is measured over all spots visible in a given timeinterval. Spot trajectories for spots starting at different radialdistances from the origin are shifted in time so that the start time forspots starting closer to the origin is replaced by the time at whichspots starting farther from the origin reach the starting locations ofthe spots closer to the origin.

In the regime illustrated in FIG. 4C, there was good agreement betweenthe calculated and observed spot trajectories.

For the DNA used in the prototype, D was measured experimentally to be2×10⁻¹² m²/s. μ_(s) was measured to have a value of approximately 1×10⁻³l/s. Using these values, the limiting spot size was calculated to be onthe order of 100 μm. Spot radii on the order of 150 to 250 μm have beenachieved in experiments.

In another experiment, a homogeneous solution of 400 ng/ml λ DNA in 1%agarose gel (0.01×TAE) was subjected to scodaphoresis. The gel wasprepared by mixing 3 ml of 1% agarose gel with 1.5 μl of 500 ng/μl48,502 bp λ DNA and 1.5 μg ethidium bromide (500 ng/ml finalconcentration). The gel was allowed to cool to approximately 65° C. andthen poured into the gel boat. The gel was arranged in a cross shape, asshown in FIG. 3C. Platinum electrodes 19 0.03 mm in diameter werelocated in open electrode regions 20 of the apparatus. The electroderegions were free from gel and filled with 0.01×TAE buffer.

The distance between opposing electrodes was approximately 2.4 cm. Afterapproximately 90 minutes, the λ DNA was found to have been concentratedin a region 21 in the center of the gel boat in a spot having a fullwidth at half maximum of about 300 μm. The concentration of the λ DNA inthe spot was enhanced by a factor of approximately 3000 to 4000 ascompared to the initial concentration of λ DNA in the gel boat. Theability to cause DNA to be concentrated in an area 21 which is away fromelectrodes is advantageous in various applications.

The concentration factor, F, that can be achieved using a square gelslab having sides of length L is calculated to be approximately:

$\begin{matrix}{F = {\frac{1}{\pi}\left( {\frac{L}{200}{\mu m}} \right)^{2}}} & (22)\end{matrix}$

Therefore, other factors being equal, increasing the dimensions of thegel slab can increase the concentration factor. For example,calculations suggest that a 35 cm×35 cm square gel slab could produce aconcentration factor on the order of 10⁶. To achieve the bestconcentration it may be desirable to take steps to inhibit diffusion ofparticles out of the 2D surface in which SCODA is being used toconcentrate the particles.

Electrophoretic SCODA in two dimensions can be performed convenientlyusing four electrodes arranged in two opposing pairs, as describedabove. Other arrangements of three or more electrodes that are notcollinear with one another could also be used. For example SCODA couldbe performed using three electrodes arranged at corners of a triangle.SCODA could also be performed using five or more electrodes arrangedaround a region of a medium.

Since the passage of electrical current through a medium can lead toheating of the medium and most practical media are electricallyconducting to some degree it is desirable to design SCODA apparatus tominimize heating, where practical, and to ameliorate the effects ofheating, where necessary. For example, SCODA may be practiced in wayswhich include one or more of:

cooling the medium through the use of a cooler in physical contact withthe medium, cooling a buffer circulating around the medium, blowing coolair over the medium or evaporatively cooling the medium;

making the medium very thin, thereby reducing the electrical currentflowing in the medium and improving dissipation of heat from the medium;

placing the medium on a thermally-conductive substrate that acts as aheat sink;

reducing the electrical conductivity of the medium by way of a chemicaltreatment or by separating from the medium unneeded species that giverise to increased electrical conductivity;

providing a reservoir of buffer and replenishing buffer surrounding themedium as the buffer evaporates (see, for example, FIG. 3B);

providing one or more temperature sensors that monitor temperature ofthe medium and controlling the temperature of the medium to remainwithin an acceptable range by controlling the electrical currentsupplied to electrodes; and,

using a driving field other than an electrical field.

Example 2: 3D SCODA

FIG. 3D shows apparatus similar to that of FIG. 3A that has beenmodified by the provision of additional Z electrodes 22A and 22B. Zelectrodes 22A and 22B are each maintained at a DC voltage. Fornegatively charged particles, Z electrodes 22A and 22B are kept morenegative in potential than the 2D SCODA electrodes 12A, 12B, 12C, and12D. The provision of the Z electrodes provides a focusing force in theZ axis, and a de-focusing force in the XY plane of medium 11. Thedefocusing force is counteracted by SCODA.

Example 3: 3D SCODA

FIG. 3E shows apparatus 24 according to an embodiment of the inventionthat provides 3D concentration of particles in a cube-shaped block ofmedium 11 by alternately performing SCODA using electrodes in XY, XZ,and YZ planes. For example, electrodes 25A, 25B, 25C, and 25D are usedfor concentration in the XY plane. Electrodes 25A, 25E, 25C and anotherelectrode (not visible in FIG. 3E) on the side of medium 11 opposed toelectrode 25E are used for concentration in the YZ plane. Electrodes25B, 25E, 25D and the electrode opposed to electrode 25E are used forconcentration in the XZ plane.

Example 4: Size Selection by SCODA

If desired, SCODA processes can be made to select DNA and similarparticles by size. This may be achieved by suitably adjusting thediffusion coefficient, D (D can be controlled by choice of medium), andthe frequency of the driving field. Using higher driving fieldfrequencies can cause larger particles to be less likely to beconcentrated by SCODA. For example, in one experiment applying a drivingfield having a period of 12 seconds was found to concentrate both long λDNA and shorter DNA fragments from a 1 kB ladder. It was found thatreducing the period of the driving field to approximately 10 ms resultedin concentration of only the shorter DNA fragments but not the longer λDNA fragments. While the inventors do not wish to be bound by anyparticular theory of operation, this size selection may be due to the 10ms period being shorter than the relaxation time for the larger λ DNAfragments and longer than the relaxation time for the shorter DNAfragments.

In the same experiment it was found that SCODA (under these conditions)did not concentrate shorter DNA fragments (smaller than a few hundredbp). The selection out of the small sizes may be due to the smallerfragments having higher values for the diffusion coefficient D.

It is believed that SCODA provides a method for separating supercoiledplasmids from plasmids that are nicked or otherwise degraded.

Example 5: Purification of DNA

Because SCODA can be made selective for different kinds of particles bychoosing a suitable medium and/or combination of driving andmobility-varying fields, SCODA can be used to purify materials, such asDNA. SCODA can be applied to cause DNA (or optionally DNA having aparticular size range) to concentrate at a spot or along a line whileother materials are not concentrated at the spot or line.

For example, in initial experiments, λ DNA was concentrated from amixture of λ DNA and bovine serum albumin (BSA). There was a 10:1concentration ratio of BSA to λ DNA. The λ DNA was concentrated into aspot, as described above. The BSA was not concentrated in the spot.

In some embodiments of the invention, denaturing agents, protease,nuclease inhibitors and/or RNAase are added to a mixture of materialsfrom which the particles are to be separated. Such agents may beprovides to facilitate one or more of:

-   -   reducing the binding of undesired molecules to fragments of DNA        or other molecules that are desired to be concentrated;    -   reducing the amount of RNA present, if so desired;    -   preventing damage to DNA; and/or    -   breaking down the undesired molecules into components that will        not be concentrated by SCODA.

In some cases it may be desirable to use SCODA to separate particles ofinterest from a mixture which includes materials, such as salts, thatcause the medium a high electrical conductivity. For example, bacterialcell cultures are often grown in media having salt contents on the orderof up to 0.4M. In cases where it is desired to use electrophoretic SCODAto separate DNA directly from a cell culture, such as an E. coliculture, the high electrical conductivity will result in higherelectrical currents in the medium. This in turn can lead to heating ofthe medium. This issue may be addressed by one or some combination ofthe heating control techniques discussed above.

Example 6: SCODA with Selective Media

The mobility of a particle in a medium may be made dependent upon thepresence in the particle of a specific DNA sequence by providing amedium with which DNA interacts by binding interactions. For example, agel may be made to include DNA oligonucleotides that are complementaryto the DNA in the particles that it is desired to concentrate. Thecomplementary DNA oligonucleotides may be covalently bonded to the gel.

If the characteristic time required for the particles to bind to thecomplementary DNA oligonucleotides is t_(on) and the characteristic timerequired for the particles to dissociate from the DNA oligonucleotidesis t_(off) then the average drift velocity for a particle in the mediumis given by:

$\begin{matrix}{\overset{\_}{v} = {{\mu(E)}*E\frac{t_{on}}{t_{on} + t_{off}}}} & (23)\end{matrix}$

where μ(E) is the field-dependent particle mobility due to reptationeffects. Typically, t_(off) is determined by an Arrhenius relationshipwhile t_(on) is determined by diffusive effects. By selecting particlesto have lengths of 1000 or more nucleotides, reasonable values fort_(off) of 1 second or less can be achieved with practical values ofelectric field (for example, electric fields in the range of 100 to 200V/cm).

FIG. 5 shows estimated particle velocity as a function of electric fieldstrength for three molecules in a sieving matrix comprising covalentlybound oligonucleotides. A first one of the molecules is a perfect matchto the covalently-bound oligonucleotides, a second one of the moleculeshas a single nucleotide mismatch to the covalently-boundoligonucleotides and a third one of the molecules is non-complementaryto the covalently-bound oligonucleotides. DC velocity is shown in solidlines. The SCODA mobility μ_(s) is shown in dashed lines.

It can be seen that there are values for the electric field that resultin the SCODA mobility for particles having DNA that binds to thecovalently bound oligonucleotides being significantly greater than forother particles. At the electric field identified by line 7 the SCODAmobility for particles that perfectly complement the covalently boundoligonucleotides is 25 times greater than it is for non-complementaryand single nucleotide mismatch molecules.

Example 7: Electric Driving Field and Thermal Mobility Varying Field

A demonstration of SCODA was carried out by thermally altering the dragcoefficient of current-carrying solute ions in an electrolyte. Whenapplying an AC potential across an electrolyte solution, andsynchronously raising and lowering the temperature of the solution, anet transport of ions is expected. If the oscillation frequency of theAC potential differs from the frequency of the thermal oscillations, adetectable component of the ionic current should be present at thedifference of the two frequencies, indicating alternating (AC) transportdue to SCODA.

FIG. 6 shows apparatus 30 that may be used to explore electric-thermalscodaphoresis. Apparatus 30 comprises a chamber 32 holding an ionicsolution 33. Electrodes 34A and 34B are immersed in solution 33. Asignal generator 35 applies an electrical signal of a first frequencybetween electrodes 34A and 34B. A heater 36 is in thermal contact withsolution 33. Heater 36 is driven by a power supply 38 so that thetemperature of solution 33 is made to vary at a second frequencydifferent from the first frequency. A detector 40 such as a lock-inamplifier is connected to electrodes 39A and 39B. Detector 40 detects asignal at a frequency equal to the difference of the first and secondfrequencies.

In an experimental prototype apparatus, a microscope slide, cover slipand epoxy were used to construct a chamber holding 300 μL of 2.0M NaClsolution. Two gold wire electrodes were glued to the microscope slide 1cm apart such that they were immersed the NaCl solution. One of theelectrodes was grounded and the other connected through a 1 kΩ resistorto an AC amplifier. Nickel-Chromium Alloy wire (NIC60-015-125-25, Omega,Stamford, Conn.) was glued to one side of the microscope cover slip toallow heating of the solution.

During operation, 1.32 Å of current was pulsed to the heater in the formof a 50% duty cycle, square wave at 10 Hz. A small fan runningcontinuously was used to cool the microscope slide during off cycles ofthe heater. A 12 Hz, 3.0V_(RMS) sine wave was applied across theresistor and electrodes. These two signals were mixed and the outputdifference frequency (of 2 Hz) was fed into the reference input of alock-in amplifier (SR830 DSP, Stanford Research Systems, Sunnyvale,Calif.). To measure the periodic current resulting from SCODA, thevoltage across the 1 kΩ resistor was measured with the lock-in amplifierand the 2 Hz component was singled out for analysis. An ionic currentoscillating at 2 Hz was detected.

The driven temperature oscillation of the sample solution was measureddirectly by a thermocouple (0.005-36, Omega) glued to the microscopeslide between the electrodes. The 2 Hz component of the thermocoupleoutput was also analyzed using the lock-in amplifier.

We assume the temperature dependent change of the electrolyte'sresistance R₀ is small compared to both the 1 kΩ current-monitoringresistor and the DC resistance of the solution (R_(DC)). The voltageacross the electrodes is:

$\begin{matrix}{{V = {V_{0}{\cos\left( {\omega\; t} \right)}}},{V_{0} = {4.23V}},{\omega_{1} = \frac{2\pi}{T_{1}}},{T_{1} = {\frac{1}{12}\sec}}} & (24)\end{matrix}$

The resistance of the salt solution is R=R_(DC)+R₀ cos(ω₂t+φ) where thefrequency of the induced thermal oscillation is ω₂=2π/T₂ with T₂= 1/10s. The total current through the solution is then:

$\begin{matrix}{I_{TOT} = \frac{V_{0}{\cos\left( {\omega_{1}t} \right)}}{R_{DC} + {R_{0}{\cos\left( {{\omega_{2}t} + \varphi} \right)}}}} & (25)\end{matrix}$

Assuming R₀ is small, Equation (24) yields an expression with asinusoidal term at the difference frequency (ω₂−ω₁) whose amplitude isgiven by:

$\begin{matrix}{I = \frac{V_{0}R_{0}}{2R_{DC}^{2}}} & (26)\end{matrix}$

A current having a magnitude of 4 μA at 2 Hz was observed.

Example 8: Electric Driving Field and Optical Mobility Varying Field

In some cases, one can alter the mobility of particles that it isdesired to move by exposing the particles to radiation. In such casesone can practice scodaphoresis by controlling the application ofradiation in time with the driving field such that the average mobilityof the particles is different for the two directions of the drivingfield. For example, one could:

-   -   apply radiation while the driving field is forcing the particles        in one direction and not apply the radiation when the driving        field is forcing the particles in the opposite direction;    -   apply radiation of one wavelength or polarization when the        driving field is forcing the particles in one direction and        apply radiation of a different wavelength or polarization when        the driving field is forcing the particles in the opposite        direction;    -   apply radiation of one intensity while the driving field is        forcing the particles in one direction and apply radiation of a        reduced intensity when the driving field is forcing the        particles in the opposite direction;    -   apply radiation having a time-varying intensity g(t) that has a        non-zero correlation with the driving field;    -   and so on.

In some cases it is not practical or desirable to use radiation to alterthe mobility of the particles themselves but it is practical to bind tothe particles other molecules that have mobilities that can becontrolled by applying radiation. The other molecules may, for example,have conformations that can be changed by applying radiation or may bindto the medium in a manner that can be controlled by applying radiation.

In some embodiments, azo-benzene is attached to particles to besubjected to scodaphoresis. Azo-benzene can isomerize from the trans- tocis-form upon exposure to UV light (300-400 nm). The azo-benzene revertsto its trans-form when it is exposed to light having a wavelengthgreater than 400 nm. In some embodiments, spiro-pyrans are attached tothe particles. Exposure of the ‘closed’ form of spiro-pyrans to UV lightinduces isomerization to yield an ‘open’ colored merocyanine species.The spiro-pyrans reverts to its ‘closed’ form on exposure to visibleradiation. The transition between these two forms is accompanied bychanges in the polar nature of the molecule.

Where radiation is used to vary the mobility of particles, differentradiation fields may be applied in different areas to achieveconcentration of the particles. For example, consider the apparatus 41shown in FIG. 7A. In apparatus 41 a medium 11 is located between twoelectrodes 42A and 42B. An AC power supply 43 applies an AC electricalsignal 44 (FIG. 7B) between electrodes 42A and 42B.

Light projectors 46A and 46B respectively illuminate portions 45A and45B of medium 11. A control 47 causes light projector 46A to illuminatearea 45A only when signal 44 creates an electrical field in a firstdirection. Control 47 causes light projector 46B to illuminate area 45Bonly when signal 44 creates an electrical field in a second directionopposed to the first direction. The result is that particles in medium11 converge on line 49 at the boundary of areas 45A and 45B from bothsides.

Many alternative constructions can be used to illuminate areas 45A and45B in time with a driving field. For example:

-   -   Light from a single lamp could be steered by a suitable optical        system to illuminate areas 45A and 45B in alternation;    -   Light from one or more lamps could be blocked from areas 45A and        45B in alternation by a suitable arrangement of mechanical or        electromechanical filters, shutters, masks or other devices        having a controllable light transmission or reflection; and,    -   so on.

Focusing in the Y direction may be achieved by rotating the lightpattern and electrical field by 90 degrees relative to medium 11.

Electrical/optical SCODA may be used to cause particles to congregate atan array of spots or along a number of lines. This can be achieved byapplying a patterned light field to the area of medium 11. This may beused to provide samples of DNA that are concentrated along spots orlines for example. Various biological applications require an array ofspots or lines of DNA.

FIGS. 8A through 8D shows a possible arrangement of four masks 50Athrough 50D that can be used to concentrate particles into an array of16 spots. Masks 50A and 50B are complementary to one another. Masks 50Cand 50D are complementary to one another. Mask 50A is applied while adriving field causes particles to move in a direction 51A. Mask 50B isapplied when the driving field causes particles to move in direction51B. It can be seen that particles will be concentrated along the fourlines 52A, 52B, 52C, and 52D if the driving field is alternated betweendirections 51A and 51B while masks 50A and 50B are applied as describedabove. Similarly, by alternately applying mask 50C with the drivingfield in direction 51C and mask 50D with the driving field in direction51D, particles will be concentrated along the four lines 53A, 53B, 53C,and 53D.

Eventually, after a number of cycles, particles will be concentrated inspots 54 at the intersections of lines 52A to 52D and lines 53A to 53Das shown in FIG. 8E. The particles may comprise, for example, desiredDNA or other molecules having attached azobenzene groups. The order inwhich masks 50A to 50D are applied (together with their correspondingdriving fields) can be varied. In simple embodiments, concentration inthe X direction is performed first using masks 50A and 50B and thenconcentration is performed in the Y direction using masks 50C and 50D.

Example 9: Optical Mobility Variation by Localized Viscosity Change

Particles to be concentrated by SCODA are located in a medium having aviscosity that varies with temperature. The mobility of the particles isdependent on the viscosity of the medium. The particles have anabsorption band. Upon absorbing radiant energy having a wavelength inthe absorption band, the particles release the absorbed energy as heat.

An alternating driving field of any suitable type is applied to theparticles. The particles are illuminated with radiation having awavelength in the absorption band and an intensity g(t). g(t) isselected so that g(t) has a non-zero correlation with the force f(t)applied to the particle by the driving field. When g(t) has a largevalue, the rate at which each particle releases thermal energy is largerthan it is when g(t) has a smaller value. The thermal energy released bythe particles in response to the absorbed radiation heats thesurrounding media and locally alters its viscosity and thus the particlemobility.

Example 10: Fluid Flow as Driving Field

A SCODA driving field may be created by causing the medium in which theparticles are situated to have a velocity that alternates in direction.For example, the medium may comprise a fluid in a pipe or capillary tubethat is caused to flow back and forth in the pipe. The mobility of theparticles may then be varied, either by causing the particles tointeract with an externally applied field or by causing the particles tointeract with a wall of the pipe in which they are located.

For example, consider a back and forth flow of a liquid in a pipe, inwhich molecules are suspended whose size is comparable to the pipediameter (e.g. large DNA in a micron size capillary). Now, vary thecapillary diameter (e.g. by providing the capillary with flexible wallssuch as walls of a silicone material and subjecting the capillary toexternal pressures) such that when the flow is in one direction, themolecules interact more frequently with the capillary wall and areretarded.

Example 11: Use of Cyclic Dilution/Concentration to Vary Mobility

Cyclic dilution/concentration may be used to vary the mobility ofparticles, especially where the particles are travelling along at ornear a surface. The concentration or viscosity of the medium in whichthe particles are travelling may be modulated over time to correlatewith the electrical or other field driving motion of the particles.

FIG. 9 shows apparatus 60 in which particles are travelling along afluid layer 62. The particles are driven by an alternating electricfield applied between electrodes 64A and 64B. A sprayer 66 dilutes fluidlayer 62 by applying a solvent when the electric field is in a firstdirection. A vacuum valve 68 is opened to cause solvent from fluid layer62 to evaporate, thereby increasing the concentration of fluid layer 62when the electric field is in a second direction. Valve 68 and sprayer66 are operated by a suitable control system (not shown).

Example 12: Pathogen Detection

In environmental sampling it is sometimes necessary to determine whethercertain pathogens are present within relatively large volumes (e.g. 1 Lor 10 L) of fluid (or solid material that can be dissolved in a fluid).Such volumes are too large for PCR to be performed in a cost effectivemanner in most cases. Filters can be used to concentrate DNA but suchfilters tend to clog. SCODA may be used to concentrate such pathogens,if present, in a sample. The sample can first be coarsely purified, thenintroduced into a medium in which particles of interest in the samplecan be concentrated by scodaphoresis. For example, the particles may beintroduced into a gel by mixing the sample with buffer and gel materialto form a large volume gel. The buffer may include detergents or otheragents to help lyse the pathogens and release their DNA into solution.

2D or 3D SCODA can them be performed to concentrate all or most of theDNA in the volume at a central location. The DNA at the central locationis contained in a volume of gel that is manageable by normal means. Theconcentrated DNA may then be PCR amplified to detect specific pathogens.DNA can optionally be extracted from the gel using, for example, acommercial kit (e.g. Qiagen™) or using the I-ZIFE extraction methodsdescribed below before performing PCR amplification. In the alternative,a piece of the gel including the concentrated DNA may be subjected toPCR. The gel tends to melt during the PCR reaction does notsignificantly adversely affect the PCR amplification in someapplications.

Example 13: Magnetic Control of Particle Mobility

FIG. 10A shows a medium 60 comprising a polymer matrix. The mediumincludes polymers 62 linked to magnetic beads 64. The magnetic beadscould be of the type currently available and used for DNA extractions. Amagnetic field generated by a suitable magnet 66 could be turned on topull magnetic beads 64 and the associated polymers 62 to one side of themedium as shown in FIG. 10B. the result is that a region 68 of themedium becomes less viscous. The magnetic particles could be released byswitching off the magnetic field to resume the situation illustrated inFIG. 10A wherein the medium in region 68 is more viscous than it is withthe magnetic field on.

The magnetic field may be patterned in two dimensions and changed overtime such that the viscosity of the medium is a function of both timeand position in the medium.

In an alternative embodiment illustrated in FIGS. 10C and 10D, theparticles being transported are themselves magnetic. The driving field,may, for example, be an electrical field. A magnetic field could beswitched on periodically to drive the particles toward a drag-inducingsurface 67. The magnetic field could be switched off to release theparticles from surface 67.

In other embodiments, the medium comprises a magneto-rheological fluidso that the medium has a viscosity that inherently varies with theapplied magnetic field.

Example 14: Acceleration as a Driving Field

A gravitationally induced flow in a density gradient may be used as adriving field. consider, for example, a tube filled with a medium, suchas a solution in which heavier or lighter particles are suspended. Thetube is located in a centrifuge so that the particles tend to traveltoward one end of the tube. The orientation of the tube is periodicallyreversed. A suitable mobility-varying field could be applied in timewith the reversals of orientation so that the particles are caused toachieve net motion in one direction along the tube.

Example 15: SCODA for Desalination

Consider an electrically insulating capillary filled with a salinesolution. If the fluid in the capillary is caused to flow then aparabolic velocity profile is established in the capillary. Fluid flowsmore quickly at the center of the capillary than near the capillarywalls. If an electric field is established across the capillary, ionswill build up preferentially within a Debye length (charge screeninglength) of the capillary walls as required to cancel the appliedelectric field. This changes the radial distribution of ions in thecapillary and thus changes the average velocity of the ions. If thefluid flow is caused to reverse periodically and the electric field isapplied only for one direction of flow then there will be a nettransport of ions in one direction along the capillary until the SCODAinduced drift is counteracted by diffusion from the accumulated iondensity gradient along the capillary.

By applying a slight DC bias to the AC fluid flow in a directionopposite to the direction of ion transport, the fluid emerging from thecapillary will have a reduced ion content.

Some Possible Variations to SCODA Methods and Apparatus

As described in Example 6 above, where the driving field and mobilityvarying field are not synchronized with one another, the result is thatthere is a flow of particles back and forth between two locations as therelative phases of the driving and mobility-varying fields vary. Thisability to move particles back and forth between two locations at acontrollable frequency may be useful in various contexts.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof. For example:

-   -   It is not necessary to generate one of the driving and        mobility-varying fields. A suitable existing field, which could        comprise a field already present for some other purpose or even        noise could be used for one of f(t) or g(t). This existing field        can be detected and a second field may be applied in time with        the detected field so that the mobility of particles is altered        in time with a driving field to produce a net drift.    -   Physically rotating electrodes at constant voltage could be used        to simulate the rotating field used for 2D SCODA.    -   Small DC biases can be used to shift the position of focused        spots.    -   Some embodiments of the invention provide wells in the medium at        locations where particles are expected to be concentrated by        SCODA. The wells may be filled with a suitable buffer solution.        Particles can diffuse into the wells as a result of SCODA        induced concentration gradient. Particles can be extracted from        the wells with a pipette or other transfer device.

Example 16: Particle Extraction Methods and Apparatus

Various methods for moving and/or concentrating particles are describedabove. Often, after particles have been concentrated, it is desirable toremove the particles from the medium in which they have beenconcentrated. For example, where DNA is concentrated in a gel, it isoften desirable to extract the DNA from the gel for subsequentprocessing.

The following description explains methods and apparatus which may beused to extract particles from a medium. These methods and apparatus maybe called “interface zero integrated field electrophoresis” (“IZIFE”)methods and apparatus. IZIFE may be used to extract particles that havebeen moved to a location and/or concentrated by a SCODA method IZIFEalso has more general application in extracting particles from media.

IZIFE exploits differences in the mobility properties of particles indifferent media (such as a gel medium and a buffer medium). Some chargedparticles (such as molecules of DNA) exhibit an electrophoretic mobilityin gel solution (such as agarose gel) that depends on the magnitude ofthe electric field applied. Such particles can be caused to drift in onedirection in such media by applying an electric field that variesasymmetrically with time. However, when those particles are in buffer orfree solution, they have an electrophoretic mobility which is constantor at least has a much lower dependence on electric field strength.Therefore, the particles stop drifting if they are carried into a mediumwhere they have a mobility that does not vary with applied field.

Application of ZIFE (zero-integrated-field-electrophoresis) to a gelcontaining charged particles will cause the particles to drift in thedirection that yields the greater mobility. If the particles enter aregion containing a buffer or free solution, they will stop drifting.Continued application of a zero time-averaged electric field causes nonet drift on the particles in the buffer solution. Therefore, theparticles tend to become concentrated in the buffer solution adjacent tothe interface between the buffer solution and the gel.

The extraction methods detailed herein permit particles to be extractedfrom a medium. The invention may be applied to extracting chargedbiopolymers such as DNA, RNA and polypeptides from electrophoresismedia, for example. Some embodiments of the invention use ZIFE to moveparticles from a medium, such as an electrophoresis gel, into anadjacent fluid. ZIFE is a form of Alternating Current (AC)electrophoresis where, the polarity of an applied electric fieldreverses periodically and the time-averaged electric field is zero. Theintensity of the electric field is greater in one polarity than in theother.

FIG. 11 is a graphical illustration of an exemplary electric field pulseused in ZIFE. As shown in FIG. 11, the pulse comprises an electric fieldE₁ applied in the positive, or “forward”, direction for a time t₁,followed by an electric field E₂ applied in the negative, or “reverse”,direction for a time t₂. If E₂=−E₁/r_(∈) (where r_(∈) is the fieldratio), and t₂=t₁r_(∈), then the time-averaged electric field is zero.The time-averaged electric field is graphically represented by theshaded areas in FIG. 11. The “positive” shaded areas (corresponding toE₁) cancel the “negative” shaded areas (corresponding to E₂). Overallthere is a zero net electric field. If the time-averaged electric fieldis exactly zero, then the ZIFE process is unbiased. If the time-averagedelectric field deviates from zero, then the ZIFE process is biased.

As discussed above, the velocity v of a particle moving in a localelectric field of amplitude E and having an electrophoretic mobility μis given by:v=μE  (27)

For linear systems, μ is constant. Particles having constantelectrophoretic mobility have no net migration in a medium (i.e. theirnet velocity is zero) when ZIFE is applied to the medium. By contrast,in non-linear systems, particles have an electrophoretic mobility thatis dependent on electric field amplitude. In such non-linear systems,there is a net migration of the particles in the direction that yieldsthe greater mobility. In such a non-linear system, the particle velocitymay be given by:v=μ(E)E  (28)

Suppose that charged particles in a medium have a field-dependentelectrophoretic mobility of the form:μ(E)=μ₀ +kE  (29)

It can be seen that the mobility of these particles increases with theamplitude of the electric field E. The distance d traveled by theparticles under the influence of a constant electric field E is given byd=vt. If an electric field pulse of the form shown in FIG. 11 isapplied, the particles will travel a greater distance during t₁ (whilethe pulse has the greater field amplitude) than the distance traveledduring t₂. This can be shown by applying Equations (27) and (29) to thedistance traveled by the particles. Hence, there is a net drift ofparticles in the “forward” direction, i.e. the direction in which theelectric field of amplitude E₁ is applied.

This net drift behavior has been demonstrated by DNA molecules inagarose gels. In such gels, DNA molecules have an electrophoreticmobility of the form given by Equation (28). The field dependence ofmobility arises from interactions between the DNA molecules and the gel.Therefore, ZIFE can be applied to DNA in an agarose gel to directparticles made up of DNA in a desired direction.

By contrast, application of ZIFE to DNA molecules in a buffer or freesolution does not produce a net migration of DNA. This is because themobility of DNA molecules in buffer solution is not field dependent. Thedifferences in mobility properties of DNA in two media (e.g. a bufferand a gel) can be exploited to move particles from within one mediuminto another medium where the particles can be accumulated. This can bedone by applying a ZIFE field across an interface between the two media.

Consider, for example, applying a ZIFE field across an interface betweena gel in which there are DNA molecules and a buffer solution. ApplyingZIFE to the molecules of DNA in the gel causes the molecules to migratein the gel toward the gel-buffer interface. Once those molecules enterthe buffer, the molecules will stop migrating. The ZIFE field may have asmall bias in the direction which tends to move the molecules from thebuffer toward the gel. This bias tends to prevent the molecules fromdiffusing too far away from the interface after they enter the buffer.The bias may prevent the molecules from encountering the electrode usedto create the ZIFE field. The bias is small enough that the particles inthe gel continue to move toward the interface (i.e. the ZIFE velocity isnot overcome by the net drift resulting from the bias).

Apparatus according to one embodiment of the invention is shown in FIGS.12A and 12B. FIG. 12A shows molecules of DNA in a gel, prior toextraction, and FIG. 12B shows molecules of DNA concentrated in abuffer, after extraction from the gel. An extraction apparatus 200comprises a gel boat 120 (which may be shaped as a rectangular box)containing a gel 122, such as agarose gel. Gel 122 fills a substantialvolume of gel boat 120. Preferably gel 122 is separated from each ofelectrodes 130B by a buffer solution in a reservoir 124. Reservoirs 124are separated from one another so that the buffer does not provide shortcircuit paths between electrodes 130B.

As shown in FIG. 12A, prior to extraction, molecules of DNA 128A areconcentrated in a column in gel 122. Molecules 128A are typically notconcentrated in such form when left in their natural state. Prior tobeing concentrated, molecules 128A are typically distributed throughoutgel 122. Molecules 128A may be concentrated into a column as shown inFIG. 12A through the use of SCODA, as described above. In thealternative, molecules 128A may be concentrated by another method. Forexample, the molecules to be extracted may be the molecules of a band ofDNA separated by conventional DC electrophoresis or PFGE

Concentration of molecules 128A in a region of gel 122 is not requiredprior to extraction. However, concentration is preferable to facilitatemore efficient extraction of the molecules.

A capillary 125 containing a small amount of buffer solution is insertedinto gel 122 so as to surround the molecules 128A to be extracted.Capillary 125 may be inserted by a robotic device which permits thelocation of insertion to be carefully controlled and which inserts thecapillary with minimal disturbance of the gel. The robotic device maycomprise a multi-axis positioner, such as an X-Y positioner, thatpositions capillary 125 over a desired location in a medium and thenlongitudinally extends the capillary into the medium. After capillary125 is inserted into gel 122, the top portion of capillary 125 containsbuffer solution, while the bottom portion of capillary 125 contains gel122. The buffer solution in capillary 125 provides an extractionreservoir 126 adjacent to gel 122. Extraction reservoir 126 meets gel122 at a buffer-gel boundary 121. The arrangement of buffer and gel incapillary 125 forms a buffer-gel interface 131. A pipette 129 isprovided above capillary 125 to suction molecules 128A after they havemigrated into extraction reservoir 126.

To provide the electric fields required for electrophoresis, anelectrode 130A is located near the tip of pipette 129. Electrode 130A ispreferably located sufficiently far from the interface that theextracted molecules do not encounter electrode 130A while the ZIFE fieldis being applied. A plurality of electrodes 130B are located in bufferreservoir 124. The electrodes may be made of platinum, for example. Moreelectrodes may be provided than those shown in FIGS. 12A and 12B.

The tip of pipette 129 is filled with a small amount of buffer so as toprovide conductivity between electrodes 130A and 130B when the pipetteis inserted in capillary 125. In one embodiment, electrodes 130B areganged to a fixed common potential (for example, electrodes 130B may begrounded), while electrode 130A is set to a different potential. Avarying electric field can be applied across buffer-gel interface 131 byvarying the potential of electrode 130A.

To perform Interface-ZIFE, a zero time-averaged pulsed electric field isapplied across buffer-gel interface 131. The pulsed electric field maybe of the form shown in FIG. 11, for example. To cause molecules 128A tomigrate in the desired direction (i.e. toward extraction reservoir 126),an electric field having an amplitude E₁ is applied in the directiontoward extraction reservoir 126, while an electric field having anamplitude E₂ is applied in the opposite direction. E₁ and E₂ are chosensuch that the particles to be extracted have a greater mobility underthe influence of E₁ than they do under the influence of E₂. For typicalmolecules and media E₁>E₂. The polarity is selected so that theparticles are driven toward interface 131 under the influence of E₁.

Application of Interface-ZIFE across buffer-gel interface 131 will causemolecules 128A in gel 122 to drift toward extraction reservoir 126.After some time, some of the molecules 128A will cross buffer-gelboundary 121 and enter into the buffer in extraction reservoir 126. Oncethese molecules reach extraction reservoir 126, Interface-ZIFE has nonet drift effect on the molecules and the molecules thus stop drifting.Eventually all (or most) of molecules 128A will cross the buffer-gelboundary 121 and migrate into extraction reservoir 126. Molecules 128Abecome concentrated in the buffer adjacent the interface.

FIG. 12B shows molecules 128B (corresponding to molecules 128A in FIG.12A) that have migrated from gel 122 into extraction reservoir 126.Thus, Interface-ZIFE can be used to collect and concentrate molecules128B in extraction reservoir 126. Pipette 129 or another device can thensuction molecules 128B from extraction reservoir 126, thereby completingthe extraction process.

FIG. 13 shows a glass capillary in an extraction experiment in which DNAmixed with a liquid gel was allowed to set within a capillary tube.Buffer was added to an upper portion of the capillary. The techniquesdescribed above were used to extract the DNA. An image of the capillarywas captured at various times (0 minutes, 60 minutes, 120 minutes) toshow the effects of Interface-ZIFE applied to a buffer-gel interface.The buffer is a TAE (Tris-Acetate-EDTA) buffer and the gel is an agarosegel containing DNA. To perform this experiment, 100 μL of liquid 1%agarose gel, mixed with 5 μL λ DNA and 2.5 μg EtBr, was pipetted intothe lower portion of a 2.5 mm inner diameter glass capillary and allowedto solidify. The upper portion of the capillary was filled withapproximately 50 μL of 0.1×TAE buffer and a first platinum electrode wasinserted into the buffer. The bottom of the capillary was then submergedin a shallow reservoir of 0.1×TAE buffer with a second platinumelectrode.

Interface-ZIFE was performed with these conditions: periodically, avoltage V₁=200 V was applied to the first electrode for a time t₁=8 s,then a voltage V₂=−100 V was applied to the second electrode for t₂=16s. The electric field was pulsed for 2 hours. The electrodes wereseparated by 5 cm. Over the course of the experiment, the upper half ofthe capillary remained filled with buffer and the DNA remained in arelatively small volume (approximately 20 μL). As shown by the images ofthe capillary, there is a progressive migration of DNA through a gel andconcentration of the DNA in a small amount of buffer above the gel.

If extraction reservoir 126 is sufficiently small, then molecules 128Bthat are concentrated in a region in extraction reservoir 126 will leavetheir concentrated region only by diffusion, which is slow over longdistances. Convective mixing of molecules 128B and extraction buffer 126should be minimized to maintain molecules 128B in their concentratedregion. To minimize convective mixing, capillary 125 should preferablyhave a small diameter. Moreover, extraction buffer 126 and gel 122 arepreferably kept at the same temperature.

In one embodiment, pipette 129 comprises a mechanized pipetter withbuilt-in electrode 130A. The mechanized pipetter aspirates buffer into adisposable pipette tip, then partially dispenses the buffer to cover thegel inside capillary 125 so that there is conductivity betweenelectrodes 130A and 130B. Computer monitoring may be used to monitor thecurrent between electrodes 130A and 130B during extraction, and detectsuch problems as bubbles or evaporation that may create an open circuitbetween the electrodes. After extraction is complete, the remainingbuffer in the pipette tip is disposed of, and the pipette tip may returnto capillary 25 to extract further samples of particles. Mechanizedpipetting may reduce unnecessary pipette tip motion so that there isminimal mixing of the concentrated particles with the surroundingbuffer. This minimizes the extraction volume and hence increases finalconcentration of the particles to be extracted.

In another embodiment, instead of inserting a capillary filled withbuffer into the gel, the gel may be cast with a cavity. The cavity isfilled with a buffer solution, and a pipette having an electrode isinserted into the buffer. The cavity functions similarly to thecapillary in collecting the particles for extraction. Molecules may becaused to enter such a cavity from the surrounding medium by generatinga concentration gradient between the medium and the cavity by SCODA.

Interface-ZIFE extraction of DNA mixtures from gels may be applied toselectively extract DNA fragments according to their size. If cycletimes t₁ and t₂ for the electric field pulse are chosen to besufficiently small, the relaxation or re-orientation time of the DNAmolecules becomes significant and introduces a length-dependence in themigration velocity of the molecules. FIG. 14 is a graph illustrating theDNA fragment velocity during an experiment as a function of fragmentlength and cycle times t₁ and t₂. In that experiment, DNA fragments ofdifferent lengths were linearly separated using standard DCelectrophoresis in a 1% agarose gel (0.1×TAE). ZIFE was then applied (ina direction perpendicular to that in which the DC electrophoresis wasperformed) to observe non-linear velocity of the fragments.

FIG. 15 shows a comparison between a DNA fragment mix and the fragmentdistribution of the same mix, after Interface-ZIFE extraction. The mixcomprised 2 μL λ DNA (48 kb, 500 ng/μL) and 4 μL 1 kb DNA ladder (0.5-10kb, 500 ng/μL) and was run in 100 μL of 1% agarose gel applying theInterface-ZIFE extraction method described above. A pulsed electricfield was applied, generated by a voltage V₁=200V applied to theelectrode in the pipette for a time of t₁=25 ms, which alternated with avoltage V₂=−100V applied to the electrodes in the gel for a time oft₂=50 ms. The pulsed electric field was applied for 3 hours. Thisprocess extracted DNA into 0.1×TAE buffer which was mixed with loadingdye and inserted into the well of a 1% agarose gel, along with a controlfrom the original mix, for standard DC electrophoresis. The λ DNA bandand short (less than 1 kb) fragments were not extracted from the gel.The size selection of Interface-ZIFE may be applied to longer fragments(100-200 kb) as well.

Parameters that can be varied to optimize extraction speed, extractionefficiency and DNA fragment length selectivity, include: magnitude ofthe electric pulsed field; frequency (cycle times) of the electricpulsed field; composition of the buffer in extraction reservoir 126;composition of gel 122; operating temperature; and the degree ofconcentration of molecules 128A.

The methods and apparatus disclosed herein may be applied for extractingcharged particles from a medium where the particles are concentrated ina particular region of the medium (such as DNA molecules concentrated ina column or pillar in gel). However, the methods and apparatus are notlimited to such application. They may also be employed to extractcharged particles that are uniformly dispersed in the medium, located orconcentrated in particular regions or bands, or otherwise distributed inthe medium. Using the methods and apparatus disclosed herein, chargedparticles, and in particular biopolymers (for example, DNA, RNA andpolypeptides), may be extracted from acrylamide, linear poly-acrylamide,POP (Perkin Elmer), agarose gels, entangled liquid solutions ofpolymers, viscous or dense solutions, solutions of polymers designed tobind specifically to the molecules whose motion is being directed,simple aqueous solutions, and the like. Interface-ZIFE used inconjunction with SCODA-based electrophoresis (for concentrating the DNAin a region) can be used to extract bacterial artificial chromosomes,plasmids and high molecular weight or genomic DNA.

IZIFE can be used to extract only selected particles from a medium.Particles having velocities that depend only linearly on the magnitudeof an applied driving field will simply oscillate back and forth whenexposed to an IZIFE driving field. Such particles will therefore remainin the medium while other particles having velocities having anon-linear dependence on applied field can be extracted from the medium.In some cases the IZIFE driving field can be constructed so thatdifferent particle species drift toward the second medium at differentrates. The concentration of the different species at the interfacebetween the media will therefore vary over time. A species which has ahigh net drift velocity under IZIFE will be extracted from the firstmedium before a species which has a lower net drift velocity.

By terminating IZIFE before slower species have been extracted from thefirst medium, the relative concentration of species having faster netdrift velocities can be increased. By removing faster species that haveaccumulated at the interface before slower drifting species have arrivedat the interface, one can increase the relative concentration at theinterface of species having slower net drift velocities under IZIFE

Example 17: Some Possible Variant Particle Extraction Methods andApparatus

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof, including but not limited to the following:

-   -   The extraction of uncharged, or electrically neutral, molecules        may be accomplished using the methods and apparatus disclosed        herein if those molecules are carried by charged molecules. For        example, neutral proteins that interact with charged micelles        may be extracted electrophoretically through their interaction        with the micelles.    -   The waveform used for implementing ZIFE may be biased in one        direction or the other. Biased ZIFE may facilitate selective        separation of the particles according to their size.

Instead of using IZIFE to extract particles from a first medium into asecond medium, one could use SCODA to extract particles from the firstmedium into a second medium. In some such embodiments a SCODA drivingfield that alternates in direction is directed across an interfacebetween the first and second media. The SCODA driving field may, forexample, be directed substantially perpendicularly to the interface. ASCODA mobility-varying field may be selected such that themobility-varying field affects the mobility of the particles in thefirst medium so as to cause the particles in the first medium to travelin a direction toward the second medium. The mobility-varying field maybe selected to affect the mobility of the particles the second medium toa degree substantially less than it affects the mobility of theparticles in the first medium. In the best case, the mobility-varyingfield does not affect the mobility of particles in the second medium. Inthis example, the SCODA effect causes particles to be transported fromthe first medium into the second medium where the particles becomeconcentrated at the interface between the first and second media. In analternative embodiment, the mobility-varying field is applied only tothose particles that are within the first medium so that the particlesdrift by SCODA into the second medium and become concentrated in thesecond medium.

Example 18: Separation of Differentially Modified Molecules

In some embodiments, molecules that are identical except for thepresence or absence of a chemical modification that alters the bindingaffinity of the molecule for a probe are separated using affinity SCODA.Some embodiments of affinity SCODA are sufficiently sensitive toseparate two molecules that have only a small difference in bindingaffinity for the immobilized affinity agent. Examples of such moleculesinclude differentially modified molecules, such as methylated andunmethylated nucleic acids, methylated or acetylated proteins, or thelike.

For example, it has been previously shown that methylation of cytosineresidues increases the binding energy of hybridization relative tounmethylated DNA sequences. RNA sequences would be expected to display asimilar increase in the binding energy of hybridization when methylatedas compared to unmethylated sequences. The inventors have shown that oneembodiment of affinity SCODA can be used to separate nucleic acidsequences differing only by the presence of a single methylated cytosineresidue. Other chemical modifications would be expected to alter thebinding energy of a nucleic acid and its complimentary sequence in asimilar manner. Modification of proteins, such as through methylation,can also alter the binding affinity of a protein of interest with aprotein, RNA or DNA aptamer, antibody, or other molecule that binds tothe protein at or near the methylation site. Accordingly, embodiments ofaffinity SCODA can be used to separate differentially modified moleculesof interest. While the examples herein are directed to methylationenrichment, affinity SCODA can also be applied to enrichment andselection of molecules with other chemical differences, including, e.g.,acetylation.

Affinity SCODA, and sequence-specific SCODA, may be used to enrich aspecific sequence of methylated DNA out of a background of methylatedand unmethylated DNA. In this application of affinity SCODA, thestrength of the SCODA focusing force may be related to the bindingenergy of the target DNA to the bound oligonucleotides. Target moleculeswith a higher binding energy may be made to focus more strongly thantargets with lower binding energy. Methylation of DNA has previouslybeen documented to slightly increase the binding energy of target DNA toits complementary sequence. Small changes in binding energy of acomplementary oligonucleotide may be exploited through affinity SCODA topreferentially enrich for methylated DNA. SCODA operating conditions maybe chosen, for example as described above, such that the methylated DNAis concentrated while unmethylated DNA of the same sequence is washedoff the gel.

Some embodiments can separate molecules with a difference in bindingenergy to an immobilized affinity agent of less than kT, the thermalexcitation energy of the target molecules. Some embodiments can separatemolecules with a difference in binding energy to an immobilized affinityagent of less than 0.19 kcal/mol. Some embodiments can separatemolecules with a difference in binding energy to an immobilized affinityagent of less than 2.6 kcal/mol. Some embodiments can separate moleculeswith a difference in binding energy to an immobilized affinity agent ofless than 3.8 kcal/mol. Some embodiments can separate molecules thatdiffer only by the presence of a methyl group. Some embodiments canseparate nucleic acid sequences that differ in sequence at only onebase.

Example 19: Mobility of a Target in an Affinity Matrix

The interactions between a target and immobilized probes in an affinitymatrix can be described by first order reaction kinetics:[T]+[P]

[T . . . P]  (30)Here [T] is the target, [P] the immobilized probe, [T . . . P] theprobe-target duplex, k_(f) is the forward (hybridization) reaction rate,and k_(r) the reverse (dissociation) reaction rate. Since the mobilityof the target is zero while it is bound to the matrix, the effectivemobility of the target will be reduced by the relative amount of targetthat is immobilized on the matrix:

$\begin{matrix}{\mu_{effective} = {\mu_{0}\frac{\lbrack T\rbrack}{\lbrack T\rbrack + \left\lbrack {T\mspace{14mu}\ldots\mspace{14mu} P} \right\rbrack}}} & (31)\end{matrix}$where μ₀ is the mobility of the unbound target. Using reasonableestimates for the forward reaction rate and an immobilized probeconcentration that is significantly higher than the concentration of theunbound target, it can be assumed that the time constant forhybridization should be significantly less than one second. If theperiod of the mobility-altering field is maintained at longer than onesecond, it can be assumed for the purposes of analysis that the bindingkinetics are fast and equation (30) can be rewritten in terms ofreaction rates:

$\begin{matrix}{{{k_{f}\lbrack T\rbrack}\lbrack P\rbrack} = {k_{r}\left\lbrack {T\mspace{14mu}\ldots\mspace{14mu} P} \right\rbrack}} & (32) \\{\lbrack T\rbrack = {\frac{k_{r}}{k_{f}}\frac{\left\lbrack {T\mspace{14mu}\ldots\mspace{14mu} P} \right\rbrack}{\lbrack P\rbrack}}} & (33)\end{matrix}$

Inserting (33) into equation (31) and simplifying yields:

$\begin{matrix}{\mu_{effective} = {\mu_{0}\frac{1}{1 + {\frac{k_{f}}{k_{r}}\lbrack P\rbrack}}}} & (34)\end{matrix}$

From this result it can be seen that the mobility can be altered bymodifying either the forward or reverse reaction rates. Modification ofthe forward or reverse reaction rates can be achieved in a number ofdifferent ways, for example by adjusting the temperature, salinity, pH,concentration of denaturants, concentration of catalysts, by physicallypulling duplexes apart with an external electric field, or the like. Inone exemplary embodiment described in greater detail below, themechanism for modifying the mobility of target molecules moving throughan affinity matrix is control of the matrix temperature.

To facilitate analysis, it is helpful to make some simplifyingassumptions. First it is assumed that there are a large number ofimmobilized probes relative to target molecules. So long as this istrue, then even if a large fraction of the target molecules become boundto the probes the concentration of free probes, [P], will not changemuch and it can be assumed that [P] is constant. Also, it is assumedthat the forward reaction rate k_(f) does not depend on temperature.This not strictly true, as the forward reaction rate does depend ontemperature. Secondary structure in the immobilized probe or in thetarget molecule can result in a temperature dependent forward reactionrate. However, in embodiments operating at a temperature range near theduplex melting temperature the reverse reaction rate has an exponentialdependence on temperature and the forward reaction rate has a muchweaker temperature dependence, varying by about 30% over a range of 30°C. around the melting temperature. It is additionally assumed that thetarget sequence is free of any significant secondary structure. Althoughthis final assumption would not always be correct, it simplifies thisinitial analysis.

To determine the temperature dependence of the reverse reaction rate, anArrhenius model for unbinding kinetics is assumed. This assumption isjustified by recent work in nanopore force spectroscopy.

$\begin{matrix}{k_{r} = {A\;{\mathbb{e}}^{\frac{\Delta\; G}{k_{b}T}}}} & (35)\end{matrix}$Here A is an empirically derived constant, ΔG is the probe-targetbinding energy, k_(b) is the Boltzmann constant, and T the temperature.Inserting this into (34), rewriting the free energy ΔG as ΔH−TΔS, andcollecting constant terms allows the mobility to be rewritten as:

$\begin{matrix}{\mu_{effective} = {\mu_{0}\frac{1}{1 + {\beta\mathbb{e}}^{\frac{{{- \Delta}\; H} + {T\;\Delta\; S}}{k_{b}T}}}}} & (36)\end{matrix}$

Equation (36) describes a sigmoidal mobility temperature dependence. Theshape of this curve is shown in FIG. 16. At low temperature the mobilityis nearly zero. This is the regime where thermal excitations areinsufficient to drive target molecules off of the affinity matrix. Athigh temperature target molecules move at the unbound mobility, wherethe thermal energy is greater than the binding energy. Between these twoextremes there exists a temperature range within which a small change intemperature results in a large change in mobility. This is the operatingregime for embodiments of affinity SCODA that utilize temperature as themobility altering parameter.

In embodiments of affinity SCODA used to separate nucleic acids based onsequence, i.e. sequence-specific SCODA, this temperature range tends tolie near the melting temperature of the probe-target duplex.Furthermore, the speed of concentration is proportional to k, which is ameasure of how much the mobility changes during one SCODA cycle.Operating near the probe-target duplex melting temperature, where theslope of the mobility versus temperature curve is steepest, maximizes kfor a given temperature swing during a SCODA cycle in embodiments wheretemperature is used as the mobility altering parameter.

In some embodiments, affinity SCODA may be conducted within atemperature gradient that has a maximum amplitude during application ofSCODA focusing fields that varies within about ±20° C., within about±10° C., within about ±5° C., or within about ±2° C. of the meltingtemperature of the target molecule and the affinity agent.

It is possible to describe affinity SCODA in one dimension by replacingthe time dependent mobility of equation (30) with the temperaturedependent mobility of equation (36) and a time dependent temperature:

$\begin{matrix}{{T\left( {x,t} \right)} = {T_{m} + {{T_{a}\left( \frac{x}{L} \right)}{\sin\left( {{\omega\; t} + \phi} \right)}}}} & (37)\end{matrix}$Here, the temperature oscillates around T_(m), the probe target meltingtemperature, and T_(a) is the maximum amplitude of the temperatureoscillations at x=±L. To get an analytical expression for the driftvelocity, v_(d)=μE, as a function of temperature, a Taylor expansion ofequation (36) is performed around T_(m):

$\begin{matrix}{\mu_{effective} = {{\mu\left( T_{m} \right)} - {\frac{\mu_{b}{\beta\Delta}\; H\; e^{\frac{{{- \Delta}\; H} + {T\;\Delta\; S}}{k_{b}T_{m}}}}{k_{b}{T_{m}^{2}\left( {1 + {\beta\; e^{\frac{{{- \Delta}\; H} + {T\;\Delta\; S}}{k_{b}T_{m}}}}} \right)}^{2}}\left( {T - T_{m}} \right)} + {O\left( \left( {T - T_{m}} \right)^{2} \right)}}} & (38)\end{matrix}$which can be rewritten as:μ_(effective)=μ(T _(m))+α(T−T _(m))+O((T−T _(m))²)  (39)

Here the first term in the Taylor expansion has been collected into theconstant α. Combining (37) and (39) into an expression for the mobilityyields an expression similar to (40):

$\begin{matrix}{{\mu(t)} + {\mu\left( T_{m} \right)} + {\left( \frac{\alpha\; T_{a}x}{L} \right){\sin\left( {{\omega\; t} + \phi} \right)}}} & (40)\end{matrix}$Equation (40) can be used to determine the time averaged drift velocityfor both the one dimensional and two dimensional cases by simplyreplacing k with:

$\begin{matrix}{\frac{\alpha\; T_{a}}{L} = {\frac{\mu_{o}{\beta\Delta}\; H\; e^{\frac{{{- \Delta}\; H} + {T\;\Delta\; S}}{k_{b}T_{m}}}}{k_{b}{T_{m}^{2}\left( {1 + {\beta\; e^{\frac{{{- \Delta}\; H} + {T\;\Delta\; S}}{k_{b}T_{m}}}}} \right)}^{2}}\left( \frac{T_{a}}{L} \right)}} & (41)\end{matrix}$

The drift velocity is then given by:

$\begin{matrix}{{{\overset{\_}{v}}_{d}\left( {x,t} \right)} = {\frac{\alpha\; T_{a}x}{L}E_{0}{\cos(\phi)}}} & (42)\end{matrix}$in one dimension, and:

$\begin{matrix}{{\overset{\_}{v}}_{d} = {\frac{E_{0}\alpha\; T_{a\;}r}{2L}\left( {{{\cos(\phi)}\hat{r}} + {{\sin(\phi)}\hat{\theta}}} \right)}} & (43)\end{matrix}$in two dimensions. This result shows that if a two dimensional gel isfunctionalized with immobilized probes (i.e. an affinity matrix), thenby combining a rotating temperature gradient with a rotating dipoleelectric field, all target molecules should be forced towards a centralregion in the gel, thus concentrating a target molecule that binds tothe immobilized probes.

Example 20: Molecular Separation with Affinity SCODA

In some embodiments, affinity SCODA is used to separate two similarmolecules (e.g. the same molecule that has been differentially modified,or which differs in sequence at only one or a few locations) withdiffering binding affinities for the immobilized probe. Beginning withtwo molecular species, each with a different binding energy to theimmobilized probes, these two molecular species can be separated bysuperimposing a washing motive force over the driving and mobilityaltering fields used to produce SCODA focusing, to provide net motion ofmolecules that have a lesser binding affinity for the immobilized probe(i.e. the molecules that have a higher binding affinity for theimmobilized probe are preferentially focused during the application ofthe SCODA focusing fields). In some embodiments, the washing force is asmall applied DC force, referred to herein as a DC bias.

In the one dimensional case when a small DC force is applied as awashing or bias force, the electric field becomes:E(x,t)=E ₀ sin(ωt)+E _(b)  (44)where E_(b) is the applied DC bias. The final drift velocity hassuperimposed on the SCODA focusing velocity a constant velocityproportional to the strength of the bias field:

$\begin{matrix}{{{\overset{\_}{v}}_{d}\left( {x,t} \right)} = {{\frac{\alpha\; T_{a}x}{2L}E_{0}{\cos(\phi)}} + {{\mu\left( T_{m} \right)}E_{b}}}} & (45)\end{matrix}$

This drift velocity will tend to move the final focus location either tothe left or right depending on the direction of bias. The amount bywhich this bias moves a focus off center depends on the strength of theinteraction between the target and probe molecules. The differentialstrength of the target-probe interaction can therefore serve as amechanism to enable molecular separation of two highly similar species.

Consider two molecules that have different binding affinities for animmobilized probe. Reducing the probe-target binding energy, ΔG inequation (36), will serve to shift the mobility versus temperature curveto the left on the temperature scale as shown in FIG. 17. The mobilityof the high binding energy target is shown by the curve on the right,while the mobility of the low binding energy target is shown by thecurve on the left.

If the SCODA system in this exemplary embodiment is operated at theoptimal focusing temperature for the higher binding energy molecule,T_(m) in FIG. 17, then the mobility of the lower binding energy moleculewill be higher and will have weaker temperature dependence. In terms ofequation (45) the molecule with lower binding energy will have a largervalue of μ(T_(m)) and a smaller value of α. This means that a lowerbinding energy molecule will have a lower SCODA drift velocity and ahigher velocity under DC bias, resulting in a different final focuslocation than the high binding energy molecule as illustrated in FIG.18.

FIG. 18 shows the effect of an applied DC bias on molecules with twodifferent binding energies for the immobilized probe according to oneembodiment. The solid curve represents the drift velocity of a targetmolecule with a lower binding energy to the bound probes than themolecules represented by the dashed curve. The final focus location isthe point where the drift velocity is equal to zero. The moleculesrepresented by the solid curve have both a lower SCODA drift velocityand a higher DC velocity compared to the molecules represented by thedashed curve. When SCODA focusing is combined with a DC bias the lowerbinding energy molecules will focus further away from the unbiased focusat x=0, resulting in two separate foci, one for each molecular species.The final focus position for the high binding energy molecule isindicated by reference numeral 30. The final focus position for the lowbinding energy molecule is indicated by reference numeral 32.

The two dimensional case is the same as the one dimensional case, thesuperimposed velocity from the applied washing bias moves the finalfocus spot off center in the direction of the washing bias.

In some embodiments, if the difference in binding energies between themolecules to be separated is large enough and a sufficiently highwashing bias is applied, the low binding energy molecules can be washedoff of the affinity matrix while molecules with higher binding energyare retained in the affinity matrix, and may be captured at a focuslocation within the affinity matrix (i.e. preferentially focused)through the application of SCODA focusing fields.

Example 21: Generation of a Time Varying Temperature Gradient

Embodiments of affinity SCODA that use variations in temperature as themobility altering field may use a periodically varying temperaturegradient to produce a convergent velocity field. A periodically varyingtemperature gradient may be provided in any suitable manner, for exampleby the use of heaters or thermoelectric chillers to periodically heatand cool regions of the medium, the use of radiative heating toperiodically heat regions of the medium, the application of light orradiation to periodically heat regions of the medium, Joule heatingusing the application of an electric field to the medium, or the like.

A periodically varying temperature gradient can be established in anysuitable manner. For example, a temperature gradient may allow aparticle increased mobility (i.e. at a higher temperature) when adriving field is applied toward the focus spot than when a driving fieldis applied away from the focus spot. In some embodiments, thetemperature gradient is rotated to produce a convergent velocity fieldin conjunction with the application of a time-varying driving force.

In some embodiments, Joule heating using an electric field is used toprovide a temperature gradient. In some embodiments, the electric fieldused to provide Joule heating to provide a temperature gradient is thesame as the electric field that provides the driving field. In someembodiments, the magnitude of the electric field applied is selected toproduce a desired temperature gradient within an affinity matrix.

In some embodiments, a spatial temperature gradient is generated using aquadrupole electric field to provide the Joule heating. In some suchembodiments, a two dimensional gel with four electrodes is provided.Voltages are applied to the four electrodes such that the electric fieldin the gel is non-uniform, containing regions of high electric field(and consequently high temperature) and low electric field. The electricfield is oriented such that the regions of high electric field tend topush negatively charged molecules towards the center of the gel, whileregions of low electric field tend to push such molecules away from thecenter of the gel. In some such embodiments, the electric field thatprovides the temperature gradient through Joule heating is also theelectric field that applies a driving force to molecules in the gel.

An example of such a field pattern is illustrated in FIG. 19. Voltagesapplied at electrodes A, B, C and D in FIG. 19 are −V, 0, 0, and 0respectively. Arrows represent the velocity of a negatively chargedanalyte molecule. Color intensity represents electric field strength.The regions near electrode A have a high electric field strength, whichdecreases towards electrode C. The high field regions near electrode Atend to push negatively charged molecules towards the center of the gel,while the lower field regions near electrodes B, C, and D tend to pushnegatively charged molecules away from the center of the gel. Inembodiments in which the electric field also provides the temperaturegradient, the affinity matrix will become hotter in regions of higherfield strength due to Joule heating. Hence, regions of high electricfield strength will coincide with regions of higher temperature and thushigher mobility. Accordingly, molecules in the high electric fieldregions near electrode A will tend to move a greater distance toward thecenter of the gel, while molecules in the lower electric field regionsnear electrodes B, C, and D have a lower mobility (are at a coolertemperature) and will move only a short distance away from the center ofthe gel.

In some embodiments, the electric field pattern of FIG. 19 is rotated ina stepwise manner by rotating the voltage pattern around the fourelectrodes such that the time averaged electric field is zero as shownin FIG. 20. This rotating field will result in net migration towards thecenter of the gel for any molecule that is negatively charged and has amobility that varies with temperature. In some embodiments, the electricfield pattern is varied in a manner other than rotation, e.g. bysequentially shifting the voltage pattern by 180°, 90°, 180°, and 90°,or by randomly switching the direction of the electric field. As shownabove, the mobility of a molecule moving through an affinity matrixdepends on temperature, not electric field strength. The appliedelectric field will tend to increase the temperature of the matrixthrough Joule heating; the magnitude of the temperature rise at anygiven point in the matrix will be proportional to the square of themagnitude of the electric field.

In embodiments in which the thermal gradient is provided by Jouleheating produced by the electric field that also provides the drivingfield, the oscillations in the thermal gradient will have the sameperiod as the electric field oscillations. These oscillations can driveaffinity SCODA based concentration in a two dimensional gel.

FIG. 20 illustrates the stepwise rotation of the electric field leadingto focusing of molecules whose mobility increases with temperature orelectric field according to such an embodiment. A particle path for anegatively charged molecule is shown. After four steps the particle hasa net displacement toward the center of the gel. Molecules that do notexperience a change in mobility with changing temperature or electricfield will experience zero net motion in a zero time averaged electricfield.

Example 22: Theoretical Predictions of Focusing and Separation

In some embodiments, the electric field and subsequently the Jouleheating within an affinity SCODA gel are controlled by both the voltageapplied to the source electrodes, and the shape of the gel. For example,superimposed rotating dipole and quadrupole fields can be used to driveelectrophoretic SCODA concentration. The ratio of the strength of thesetwo fields, the dipole to quadrupole ratio (D/Q), has an impact on theefficiency of SCODA focusing with a maximum at around D/Q=4.5, howeverthe optimum is relatively flat with the SCODA force staying relativelyconstant for values between 1.75 and 10. One convenient choice of D/Q is2. With this particular choice, only two distinct potentials need to beapplied to the source electrodes, which can be achieved by connectingone electrode to a common voltage rail, grounding the other three, androtating this pattern in a stepwise manner through the four possibleconfigurations as shown in Table 2. Although analog amplifiers can beused and were used in the examples described herein, using a D/Q ratioof 2 allows one to use discrete MOSFET switches, which simplifies andreduces the required size and complexity of the power supplies.

TABLE 2 Voltage pattern for SCODA focusing with D/Q = 2. Electrode AElectrode B Electrode C Electrode D Step 1 −V 0 0 0 Step 2 0 −V 0 0 Step3 0 0 −V 0 Step 4 0 0 0 −V

A starting point for a sequence specific gel geometry was the four-sidedgel geometry used for the initial demonstration of electrophoreticSCODA. This geometry can be defined by two numbers, the gel width andthe corner radius. The inventors started by using a geometry that had awidth of 10 mm and a corner radius of 3 mm. An electro-thermal model ofthis geometry was implemented in COMSOL MULTIPHYSICS® modeling software(COMSOL, Inc, Burlington Mass., USA) to estimate the electric field andtemperature profiles within the gel and establish whether or not thosefield and temperature profiles could drive concentration of a targetwith a temperature dependent mobility. The model used simultaneouslysolves Ohm's Law and the heat equation within the domain, using thepower density calculated from the solution of Ohm's Law as the sourceterm for the heat equation and using the temperature solution from theheat equation to determine the temperature dependent electricalconductivity of the electrolyte in the gel.

To obtain an accurate estimate of the temperature profile within thegel, the heat conducted out of the top and bottom of the gel aremodeled. Boundary conditions and other model parameters are illustratedin FIG. 21. The thermal properties of water and electrical properties of0.2 M NaCl were used. The gel cassettes are placed on an aluminumspreader plate that acts as a constant temperature reservoir. To modelheat flow into the spreader plate the heat transfer coefficient of theglass bottom, given by k/t, was used. The temperature and electric fieldprofiles solved by this model for a single step of the SCODA cycle areshown in FIG. 22. The voltage applied to the four electrodes was −120 V,0 V, 0 V, 0 V, and the spreader plate temperature was set to 55° C. (328K). The color map indicates gel temperature and the vector field showsthe relative magnitude and direction of the electric field within thegel. Note that as DNA is negatively charged its migration direction willbe opposite to the direction of the electric field.

Using experimentally determined values of mobility versus temperaturefor a given molecule and the thermal model described above, it ispossible to determine the SCODA velocity everywhere in the gel for thatparticular molecule by taking the time average of the instantaneousdrift velocity integrated over one complete cycle:

$\begin{matrix}{{\overset{\rightarrow}{v}}_{s} = {\frac{1}{\tau}{\int_{0}^{\tau}{{\mu\left( {T\left( {\overset{\rightarrow}{r},t} \right)} \right)}{\overset{\rightarrow}{E}\left( {\overset{\rightarrow}{r},t} \right)}\ {dt}}}}} & (46)\end{matrix}$where μ is the temperature dependent mobility, E the electric field andτ the period of the SCODA cycle. The temperature and electric field weresolved for four steps in the SCODA cycle and coupled with the mobilityfunction in equation (36). In this manner, the SCODA velocity everywherein the gel can be calculated. Since discrete steps are being used, if itis assumed that the period is long enough that the phase lag between theelectric field and temperature can be neglected, then the integral inequation (46) becomes a sum:

$\begin{matrix}{{\overset{\rightarrow}{v}}_{s} = \frac{\sum{{\mu\left( {T_{i}\left( \overset{\rightarrow}{r} \right)} \right)}{{\overset{\rightarrow}{E}}_{i}\left( \overset{\rightarrow}{r} \right)}t_{i}}}{\sum t_{i}}} & (47)\end{matrix}$where the velocity is summed over all four steps in the cycle.

As an example, FIG. 23 shows a vector plot of the SCODA velocity usingthe experimentally determined mobility versus temperature curve for theperfect match target shown in FIG. 25 (example described below) and thetemperature and electric field values calculated above.

The velocity field plotted in FIG. 23 shows a zero velocity point at thegeometric center of the gel, with the velocity at all other points inthe gel pointing towards the center. Thus, target molecules can becollected within the gel at the center of the electric field pattern.

In embodiments that are used to separate two similar molecules based ondifferences in binding affinity for the immobilized probe, a washingforce is superimposed over the SCODA focusing fields described above. Insome embodiments, the washing force is a DC electric field, describedherein as a DC bias. For molecules having affinity to the immobilizedprobe, the SCODA focusing force applied by the SCODA focusing fieldsdescribed above will tend to counteract movement of a molecule caused bythe washing field, i.e. the SCODA focusing fields will tend to exert arestoring force on the molecules and the molecules will bepreferentially focused as compared with molecules having a smallerbinding affinity. Molecules that have a smaller binding affinity to theimmobilized probe will have a greater mobility through the affinitymatrix, and the restoring SCODA force will be weaker. As a result, thefocus spot of molecules with a smaller binding affinity will be shifted.In some cases, the restoring SCODA force will be so weak that suchmolecules with a smaller binding affinity will be washed out of theaffinity matrix altogether.

In order to enrich for a specific biomolecule from a population of othersimilar biomolecules using affinity SCODA, one may operate SCODAfocusing electric fields with a superimposed DC bias. The DC bias maymove the focused molecules off center, in such a way that the moleculeswith a lower binding energy to the immobilized binding sites movefurther off center than the molecules with higher binding energies, thuscausing the focus to split into multiple foci. For molecules withsimilar binding energies, this split may be small while washing underbias. The DC bias may be superimposed directly over the focusing fields,or a DC field may be time multiplexed with the focusing fields.

In one exemplary embodiment used to separate nucleic acids havingsimilar sequences, a DC bias is superimposed over the voltage patternshown in Table 2, resulting in the voltage pattern shown below in Table3. In some embodiments, the DC bias is applied alternately with theSCODA focusing fields, i.e. the SCODA focusing fields are applied for aperiod of time then stopped, and the DC bias is applied for a period oftime then stopped.

TABLE 3 Applied voltages for focusing under a DC bias. Shown are valuesfor a 120 V SCODA focusing potential superimposed over a 10 V DC bias.Electrode A Electrode B Electrode C Electrode D Step 1 −120 5 10 5 Step2 0 −115 10 5 Step 3 0 5 −110 5 Step 4 0 5 10 −115

The resulting velocity plots of both the perfect match and single basemismatch targets in the presence of the applied DC bias are shown inFIGS. 24A and 24B, respectively. Electric field and temperature werecalculated using COMSOL using a spreader plate temperature of 61° C.Velocity was calculated using equation (47) and the experimentallyobtained data fits shown in FIG. 25 (see description below). The zerovelocity location of the perfect match target has been moved slightlyoff center in the direction of the bias (indicated with a circularspot), however the mismatch target has no zero velocity point within thegel. These calculations show that it is possible to completely wash atarget with a smaller binding affinity from the immobilized probe fromthe gel area while capturing the target with a higher binding affinity,enabling selective purification, concentration and/or detection of aspecific sequence, even where the nucleotide targets differ in sequenceat only one position.

In some embodiments, the optimal combination of the driving field andthe mobility altering field used to perform SCODA focusing where thereis a maximum difference in focusing force between similar molecules isempirically determined by measuring the velocity of sample moleculesthrough a medium as a function of the mobility varying field. Forexample, in some embodiments the mobility of a desired target moleculeand a non-desired target molecule at various temperatures is measured inan affinity matrix as described above, and the temperature range atwhich the difference in relative mobility is greatest is selected as thetemperature range for conducting affinity SCODA. In some embodiments,the focusing force is proportional to the rate at which the velocitychanges with respect to the perturbing field dv/df, where v is themolecule velocity and f the field strength. One skilled in the art maymaximize dv/df so as to maximize SCODA focusing and to enable fastwashing of contaminants that do not focus. To maximally separate twosimilar molecules, affinity SCODA may be carried out under conditionssuch that dv_(a)/df−dv_(b)/df (where v_(a) is the velocity of moleculea, and v_(b) is the velocity of molecule b) is maximized.

In some embodiments, the strength of the electric field applied to anaffinity matrix is calculated so that the highest temperature within thegel corresponds approximately to the temperature at which the differencein binding affinity between two molecules to be separated is highest.

In some embodiments, the temperature at which the difference in bindingaffinity between the two molecules to be separated is highestcorresponds to the temperature at which the difference between themelting temperature of a target molecule and the affinity agent and themelting temperature of a non-target molecule and the affinity agent ishighest. In some embodiments, the maximum difference between the meltingtemperature of a target molecule and the affinity agent and the meltingtemperature of a non-target molecule and the affinity agent is less thanabout 9.3° C., in some embodiments less than about 7.8° C., in someembodiments less than about 5.2° C., and in some embodiments less thanabout 0.7° C.

In some embodiments, the ratio of target molecules to non-targetmolecules that can be separated by affinity SCODA is any ratio from 1:1to 1:10,000 and any value therebetween, e.g. 1:100 or 1:1,000. In someembodiments, after conducting affinity SCODA, the ratio of non-targetmolecules relative to target molecules that is located in a focus spotof the target molecules has been reduced by a factor of up to 10,000fold.

Example 22: Phase Lag Induced Rotation

In some embodiments, to separate molecules with different affinities forthe immobilized affinity agent, a DC bias is superimposed over the SCODAfocusing fields as described above. If the separation in binding energyis great enough then the mismatched target can be washed entirely off ofthe gel. The ability to wash weakly focusing contaminating fragmentsfrom the gel can be affected by the phase lag induced rotation discussedabove, where the SCODA velocity of a two dimensional system was givenby:{right arrow over (v)} _(SCODA) =|v _(SCODA)|(cos(ϕ){circumflex over(r)}+sin(ϕ){circumflex over (θ)})  (48)where ϕ is the phase lag between the electric field oscillations and themobility varying oscillations. Aside from reducing the proportion of theSCODA velocity that contributes to concentration this result hasadditional implications when washing weakly focusing contaminants out ofan affinity matrix. The rotational component will add to the DC bias andcan result in zero or low velocity points in the gel that cansignificantly increase the time required to wash mismatched targets fromthe gel.

To counteract the effects of a rotational component of motion that mayarise in embodiments in which there is a phase lag between the electricfield oscillations and the mobility varying oscillations, the directionin which the SCODA focusing fields are applied may be rotatedperiodically. In some embodiments, the direction in which the SCODAfocusing fields are rotated is altered once every period.

Example 23: Optical Feedback

In some embodiments where one molecule of interest (e.g., a targetnucleic acid) is concentrated in an affinity matrix while a second,similar, molecule (e.g., the non-target nucleic acid) is washed off ofthe affinity matrix, optical feedback may be used to determine whenwashing is complete and/or to avoid running the target out of theaffinity matrix.

The two foci of similar molecules may be close together geographically,and optical feedback may be used to ensure the molecule of interest isnot washed off the gel. For example, using a fluorescent surrogate forthe molecule of interest or the contaminating molecules (or both) onecan monitor their respective positions while focusing under bias, anduse that geographical information to adjust the bias ensuring that themolecule of interest is pushed as close to the edge of the gel aspossible but not off, while the contaminating molecule may be removedfrom the gel.

In some embodiments, the molecules to be separated are differentiallylabeled, e.g. with fluorescent tags of a different color. Real-timemonitoring using fluorescence detection can be used to determine whenthe non-target molecule has been washed off of the affinity matrix, orto determine when the foci of the target molecule and the non-targetmolecule are sufficiently far apart within the affinity matrix to allowboth foci to be separately extracted from the affinity matrix.

In some embodiments, fluorescent surrogate molecules that focussimilarly to the target and/or non-target molecules may be used toperform optical feedback. By using a fluorescent surrogate for a targetmolecule, a non-target molecule, or both a target molecule and anon-target molecule, the respective positions of the target moleculeand/or the non-target molecule can be monitored while performingaffinity focusing under a washing bias. The location of the surrogatemolecules within the affinity matrix can be used to adjust the washingbias to ensure that the molecule of interest is pushed as close to theedge of the gel as possible but not off, while the contaminatingmolecule may be washed off the gel.

In some embodiments, fluorescent surrogate molecules that focussimilarly to the target and/or non-target molecules but will not amplifyin any subsequent PCR reactions that may be conducted can be added to asample to be purified. The presence of the fluorescent surrogatemolecules within the affinity matrix enables the use of optical feedbackto control SCODA focusing conditions in real time. Fluorescencedetection can be used to visualize the position of the fluorescentsurrogate molecules in the affinity matrix. In embodiments where thefluorescent surrogate mimics the focusing behavior of the targetmolecule, the applied washing force can be decreased when thefluorescent surrogate approaches the edge of the affinity matrix, toavoid washing the target molecule out of the affinity matrix. Inembodiments where the fluorescent surrogate mimics the focusing behaviorof the non-target molecule that is to be separated from the targetmolecule, the applied washing force can be decreased or stopped afterthe fluorescent surrogate has been washed out of the affinity matrix, oralternatively when the location of the fluorescent surrogate approachesthe edge of the affinity matrix.

Example 24: Separation of Differentially Modified Molecules

In some embodiments, molecules that are identical except for thepresence or absence of a chemical modification that alters the bindingaffinity of the molecule for a probe are separated using affinity SCODA.Some embodiments of affinity SCODA are sufficiently sensitive toseparate two molecules that have only a small difference in bindingaffinity for the immobilized affinity agent. Examples of such moleculesinclude differentially modified molecules, such as methylated andunmethylated nucleic acids, methylated or acetylated proteins, or thelike.

For example, it has been previously shown that methylation of cytosineresidues increases the binding energy of hybridization relative tounmethylated DNA sequences. RNA sequences would be expected to display asimilar increase in the binding energy of hybridization when methylatedas compared with unmethylated sequences. The inventors have shown thatone embodiment of affinity SCODA can be used to separate nucleic acidsequences differing only by the presence of a single methylated cytosineresidue. Other chemical modifications would be expected to alter thebinding energy of a nucleic acid and its complimentary sequence in asimilar manner. Modification of proteins, such as through methylation,can also alter the binding affinity of a protein of interest with aprotein, RNA or DNA aptamer, antibody, or other molecule that binds tothe protein at or near the methylation site. Accordingly, embodiments ofaffinity SCODA can be used to separate differentially modified moleculesof interest. While the examples herein are directed to methylationenrichment, affinity SCODA can also be applied to enrichment andselection of molecules with other chemical differences, including e.g.acetylation.

Affinity SCODA, and sequence-specific SCODA, may be used to enrich aspecific sequence of methylated DNA out of a background of methylatedand unmethylated DNA. In this application of affinity SCODA, thestrength of the SCODA focusing force may be related to the bindingenergy of the target DNA to the bound oligonucleotides. Target moleculeswith a higher binding energy may be made to focus more strongly thantargets with lower binding energy. Methylation of DNA has previouslybeen documented to slightly increase the binding energy of target DNA toits complementary sequence. Small changes in binding energy of acomplementary oligonucleotide may be exploited through affinity SCODA topreferentially enrich for methylated DNA. SCODA operating conditions maybe chosen, for example as described above, such that the methylated DNAis concentrated while unmethylated DNA of the same sequence is washedoff the gel.

Some embodiments can separate molecules with a difference in bindingenergy to an immobilized affinity agent of less than kT, the thermalexcitation energy of the target molecules. Some embodiments can separatemolecules with a difference in binding energy to an immobilized affinityagent of less than 0.19 kcal/mol. Some embodiments can separatemolecules with a difference in binding energy to an immobilized affinityagent of less than 2.6 kcal/mol. Some embodiments can separate moleculeswith a difference in binding energy to an immobilized affinity agent ofless than 3.8 kcal/mol. Some embodiments can separate molecules thatdiffer only by the presence of a methyl group. Some embodiments canseparate nucleic acid sequences that differ in sequence at only onebase.

Example 25: Applications of Affinity SCODA

Systems and methods for separating, purifying, concentrating and/ordetecting differentially modified molecules as described above can beapplied in fields where detection of biomarkers, specific nucleotidesequences or differentially modified molecules is important, e.g.epigenetics, fetal DNA detection, pathogen detection, cancer screeningand monitoring, detection of organ failure, detection of various diseasestates, and the like. For example, in some embodiments affinity SCODA isused to separate, purify, concentrate and/or detect differentiallymethylated DNA in such fields as fetal diagnostic tests utilizingmaternal body fluids, pathogen detection in body fluids, and biomarkerdetection in body fluids for detecting cancer, organ failure, or otherdisease states and for monitoring the progression or treatment of suchconditions.

In some embodiments, a sample of bodily fluid or a tissue sample isobtained from a subject. Cells may be lysed, genomic DNA is sheared, andthe sample is subjected to affinity SCODA. In some embodiments,molecules concentrated using affinity SCODA are subjected to furtheranalysis, e.g. DNA sequencing, digital PCR, fluorescence detection, orthe like, to assay for the presence of a particular biomarker ornucleotide sequence. In some embodiments, the subject is a human.

It is known that fetal DNA is present in maternal plasma, and thatdifferential methylation of maternal versus fetal DNA obtained from thematernal plasma can be used to screen for genetic disorders (see e.g.Poon et al., 2002, Clinical Chemistry 48:1, 35-41). However, one problemthat is difficult to overcome is discrimination between fetal andmaternal DNA. Affinity SCODA as described above may be used topreferentially separate, purify, concentrate and/or detect DNA which isdifferentially methylated in fetal DNA versus maternal DNA. For example,affinity SCODA may be used to concentrate or detect DNA which ismethylated in the fetal DNA, but not in maternal DNA, or which ismethylated in maternal DNA but not fetal DNA. In some embodiments, asample of maternal plasma is obtained from a subject and subjected toaffinity SCODA using an oligonucleotide probe directed to a sequence ofinterest. The detection of two foci after the application of SCODAfocusing fields may indicate the presence of DNA which is differentiallymethylated as between the subject and the fetus. Comparison to areference sample from a subject that exhibits a particular geneticdisorder may be used to determine if the fetus may be at risk of havingthe genetic disorder. Further analysis of the sample of DNA obtainedthrough differential modification SCODA through conventional methodssuch as PCR, DNA sequencing, digital PCR, fluorescence detection, or thelike, may be used to assess the risk that the fetus may have a geneticdisorder.

One embodiment of the present systems and methods is used to detectabnormalities in fetal DNA, including chromosome copy numberabnormalities. Regions of different chromosomes that are known to bedifferentially methylated in fetal DNA as opposed to maternal DNA areconcentrated using affinity SCODA to separate fetal DNA from maternalDNA based on the differential methylation of the fetal DNA in a maternalplasma sample. Further analysis of the separated fetal DNA is conducted(for example using qPCR, DNA sequencing, fluorescent detection, or othersuitable method) to count the number of copies from each chromosome anddetermine copy number abnormalities.

Most cancers are a result of a combination of genetic changes andepigenetic changes, such as changes in DNA methylation (e.g.hypomethylation and/or hypermethylation of certain regions, see e.g.Ehrich, 2002, Oncogene 21:35, 5400-5413). Affinity SCODA can be used toseparate, purify, concentrate and/or detect DNA sequences of interest toscreen for oncogenes which are abnormally methylated. Embodiments ofaffinity SCODA are used in the detection of biomarkers involving DNAhaving a different methylation pattern in cancerous or pre-cancerouscells than in healthy cells. Detection of such biomarkers may be usefulin both early cancer screening, and in the monitoring of cancerdevelopment or treatment progress. In some embodiments, a sampleobtained from a subject, e.g. a sample of a bodily fluid such as plasmaor a biopsy, may be processed and analyzed by differential modificationSCODA using oligonucleotide probes directed to a sequence of interest.The presence of two foci during the application of SCODA fields mayindicate the presence of differential methylation at the DNA sequence ofinterest. Comparison of the sample obtained from the subject with areference sample (e.g. a sample from a healthy patient and/or a sampleknown to originate from cancerous or pre-cancerous tissue) can indicatewhether the cells of the subject are at risk of being cancerous orpre-cancerous. Further analysis of the sample of DNA obtained throughdifferential modification SCODA through conventional methods such asPCR, DNA sequencing, digital PCR, fluorescence detection, or the like,may be used to assess the risk that the sample includes cells that maybe cancerous or pre-cancerous, to assess the progression of a cancer, orto assess the effectiveness of treatment.

In some embodiments, a specific nucleotide sequence is captured in thegel regardless of methylation (i.e. without selecting for a particularmethylation status of the nucleic acid). Undesired nucleotide sequencesand/or other contaminants may be washed off the gel while the specificnucleotide sequence remains bound by oligonucleotide probes immobilizedwithin the separation medium. Then, differential methylation SCODA isused to focus the methylated version of the sequence while electricallywashing the unmethylated sequence toward a buffer chamber or another gelwhere it can then be recovered. In some embodiments, the unmethylatedsequence could be preferentially extracted.

In some embodiments, biomolecules in blood related to disease states orinfection are selectively concentrated using affinity SCODA. In someembodiments, the biomolecules are unique nucleic acids with sequence orchemical differences that render them useful biomarkers of diseasestates or infection. Following such concentration, the biomarkers can bedetected using PCR, sequencing, or similar means. In some embodiments, asample of bodily fluid or tissue is obtained from a subject, cells arelysed, genomic DNA is sheared, and affinity SCODA is performed usingoligonucleotide probes that are complimentary to a sequence of interest.Affinity SCODA is used to detect the presence of differentiallymethylated populations of the nucleic acid sequence of interest. Thepresence of differentially methylated populations of the target sequenceof interest may indicate a likelihood that the subject suffers from aparticular disease state or an infection.

In some embodiments, the focusing pattern of the target nucleic acidproduced by affinity SCODA from a subject is compared with the focusingpattern of the target nucleic acid produced by affinity SCODA from oneor more reference samples (e.g. an equivalent sample obtained from ahealthy subject, and/or an equivalent sample obtained from a subjectknown to be suffering from a particular disease). Similarities betweenthe focusing pattern produced by the sample obtained from the subjectand a reference sample obtained from a subject known to be sufferingfrom a particular disease indicate a likelihood that the subject issuffering from the same disease. Differences between the focusingpattern produced from the sample obtained from the subject and areference sample obtained from a healthy subject indicate a likelihoodthat the subject may be suffering from a disease. Differences in thefocusing pattern produced from the sample obtained from the subject anda reference sample obtained from a healthy subject may indicate thepresence of a differential modification or a mutation in the subject ascompared with the healthy subject.

Example 26: Use of Multiple Affinity Agents to Capture Multiple TargetMolecules

In some embodiments, affinity SCODA is used to separate, purify,concentrate and/or detect more than one sequence per sample. Theexamples described herein demonstrate that it is possible to concentratetarget DNA at probe concentrations as low as 1 μM, as well as with probeconcentrations as high as 100 μM. In some embodiments, multiplexedconcentration is be performed by immobilizing a plurality of differentaffinity agents in the medium to provide an affinity matrix. In someembodiments, at least two different affinity agents are immobilizedwithin a medium to separate, purify, concentrate and/or detect at leasttwo different target molecules. In some embodiments, each one of theaffinity agents is an oligonucleotide probe with a different sequence.In some embodiments, anywhere between 2 and 100 differentoligonucleotide probes are immobilized within a medium to provide anaffinity matrix, and anywhere between 2 and 100 different targetmolecules are separated, purified, concentrated and/or detectsimultaneously in a single affinity gel. Each one of the targetmolecules may be labeled with a different tag to facilitate detection,for example each one of the target molecules could be labeled with adifferent color of fluorescent tag.

In some embodiments where the binding energy between each of the two ormore affinity agents and the two or more target molecules differs, thetwo or more target molecules may be differentially separated within theaffinity matrix by the application of SCODA focusing fields at anappropriate temperature. In some embodiments, a first target moleculewith a lower melting temperature for its corresponding affinity agentmay be preferentially separated from a second target molecule with arelatively higher melting temperature for its corresponding affinityagent. In some such embodiments, the first molecule is preferentiallyconcentrated by conducting SCODA focusing at a temperature that issufficiently low that a second target molecule with a relatively highermelting temperature for its corresponding affinity agent does not focusefficiently (i.e. a temperature at which the mobility of the secondtarget molecule within the affinity matrix is relatively low), butsufficiently high to enable efficient focusing of the first molecule. Insome such embodiments, the first and second molecules are differentiallyseparated through the application of a washing bias, e.g. a DC bias, ata temperature that is sufficiently low that the second target moleculeis not displaced or is displaced only slowly by the washing bias, butsufficiently high that the first target molecule is displaced or isdisplaced at a higher velocity by the washing bias.

Example 27: Apparatus for Performing Affinity SCODA

In some embodiments, affinity SCODA is performed on an electrophoresisapparatus comprising a region for containing the affinity matrix, bufferreservoirs, power supplies capable of delivering large enough voltagesand currents to cause the desired effect, precise temperature control ofthe SCODA medium (which is a gel in some embodiments), and a two colorfluorescence imaging system for the monitoring of two differentmolecules in the SCODA medium.

Example 28: Affinity SCODA With Single Base Mismatch

To verify the predicted temperature dependent mobility expressed inequation (36), experiments were performed to measure the response oftarget DNA velocity to changes in temperature. Initial experiments weredone with 100 nucleotide oligonucleotides as target DNA.Oligonucleotides are single stranded so do not need to be denatured tointeract with the affinity gel. The oligonucleotides are alsosufficiently short that they have a negligible field dependent mobility.Longer nucleic acid molecules, e.g. greater than about 1000 nucleotidesin length, may be difficult to separate based on sequence, as longermolecules have a tendency to focus in a non-sequence-specific mannerfrom the electrophoretic SCODA effect in embodiments using Joule heatingprovided by an electric field to provide the temperature gradient.

To perform these measurements a polyacrylamide gel (4% T, 2% C) in 1×TB(89 mM tris, 89 mM boric acid) with 0.2 M NaCl and 10 μM acrydite probe(SEQ ID NO.: 1) oligo was cast in a one dimensional gel cassettecontaining only two access ports. Polymerization was initiated throughthe addition of 2 μl of 10% w/v APS and 0.2 μ1 TEMED per ml of gel.

Mobility measurements were performed on two different 100 nucleotideoligonucleotides differing by a single base containing sequences with aperfect match (PM) (SEQ ID NO.: 2) to the probe and a single basemismatch (sbMM) (SEQ ID NO.: 3). These target oligonucleotides were endlabeled with either 6-FAM or Cy5 (IDT DNA). Probe and target sequencesused for these experiments are shown in Table 4. The regions of the PMand sbMM target oligonucleotides that are complementary to theimmobilized probe are shown in darker typeface than the other portionsof these oligonucleotides. The position of the single base mismatch isunderlined in the sbMM target sequence.

TABLE 4 Probe and target oligonucleotide sequences used for sequencespecific SCODA. Sequence Probe 5′ ACT GGC CGT CGT TTT ACT 3′ (SEQ IDNO.: 1) PM Target 5′ CGA TTA AGT TGA GTA ACG CCA CTA (SEQ ID NO.: 2) TTTTCA CAG TCA TAA CCA TGT AAA ACG ACG GCC AGT GAA TTA GCG ATG CAT ACC TTGGGA TCC TCT AGA ATG TAC C 3′ sbMM Target 5′ CGA TTA AGT TGA GTA ACG CCACTA (SEQ ID NO.: 3) TTT TCA CAG TCA TAA CCA TGT AAA ACT ACG GCC AGT GAATTA GCG ATG CAT ACC TTG GGA TCC TCT AGA ATG TAC C 3′

The probe sequence was chosen to be complementary to pUC19 forsubsequent experiments with longer targets, discussed below. The 100nucleotide targets contain a sequence complementary to the probe(perfect match: PM) or with a single base mismatch (sbMM) to the probewith flanking sequences to make up the 100 nucleotide length. Theflanking sequences were designed to minimize the effects of secondarystructure and self-hybridization. Initial sequences for the regionsflanking the probe binding site were chosen at random. Folding andself-hybridization energies were then calculated using MFOLD (M. Zuker,“Mfold web server for nucleic acid folding and hybridizationprediction,”. Nucleic Acids Res. 31 (13), 3406-3415, 2003, incorporatedherein by reference), and the sequences were altered one base at a timeto minimize these effects ensuring that the dominant interactions wouldbe between target strands and the probe.

Table 5 shows the binding energies and melting temperatures for thesequences shown in Table 4 calculated using MFOLD. The binding energy,ΔG, is given as ΔH−TΔS, where ΔH is the enthalpy and ΔS the entropy inunits of kcal/mol and kcal/mol K respectively. The following parametervalues were used for calculation of the values in Table 5:temperature=50° C., [Na⁺]=0.2 M, [Mg²⁺]=0 M, strand concentration=10 μM.The largest T_(m) for non-probe-target hybridization is 23.9° C. and thegreatest secondary structure T_(m) is 38.1° C. Both of these values arefar enough below the sbMM target-probe T_(m) that they are not expectedto interfere target-probe interactions.

TABLE 5 Binding energies and melting temperatures for Table 4 sequences.Probe PM Target sbMM Target Secondary (SEQ ID NO.: 1) (SEQ ID NO.: 2)(SEQ ID NO.: 3) Structure Probe  −35.4 + 0.1012*T −145.3 + 0.4039*T −126.8 + 0.3598*T  −20.3 + 0.07049*T (SEQ ID NO.: 1) T_(m) = 12.2° C.T_(m) = 65.1° C. T_(m) = 55.8° C. T_(m) = 14.8° C. PM Target −145.3 +0.4039*T −40.2 + 0.1124*T −40.2 + 0.1111*T −24.3 + 0.07808*T (SEQ IDNO.: 2) T_(m) = 65.1° C. T_(m) = 23.9° C. T_(m) = 20.9° C. T_(m) = 38.1°C. sbMM Target −126.8 + 0.3598*T −40.2 + 0.1111*T −40.2 + 0.1124*T−24.3 + 0.07808*T (SEQ ID NO.: 3) T_(m) = 55.8° C. T_(m) = 20.9° C.T_(m) = 23.9° C. T_(m) = 38.1° C.

To measure the velocity response as a function of temperature thefluorescently labeled target was first injected into the gel at hightemperature (70° C.), and driven under a constant electric field intothe imaging area of the gel. Once the injected band was visible thetemperature of the spreader plate was dropped to 55° C. An electricfield of 25 V/cm was applied to the gel cassette while the temperaturewas ramped from 40° C. to 70° C. at a rate of 0.5° C./min. Images of thegel were taken every 20 sec. Image processing software written inLABVIEW® (National Instruments, Austin Tex.) was used to determine thelocation of the center of the band in each image and this position datawas then used to calculate velocity.

FIG. 25 shows a plot of target DNA mobility as a function oftemperature. Using the values for the probe and target sequences shownin Table 5, the velocity versus temperature curves were fit to equation(36) to determine the two free parameters: the mobility μ₀, and β aconstant that depends on the kinetics of the hybridization reaction.

A fit of the data shown in FIG. 25 shows good agreement with the theoryof migration presented above. Data for the mismatch mobility are shownas the curve on the left, and data for the perfect match mobility areshown as the curve on the right. The R² value for the PM fit and MM fitswere 0.99551 and 0.99539 respectively. The separation between theperfect match and single base mismatch targets supports that there is anoperating temperature where the focusing speed of the perfect matchtarget is significantly greater than that of the mismatched targetenabling separation of the two species through application of a DC biasfield as illustrated in FIG. 18.

Example 29: Selective Separation of Molecules Using Affinity SCODA

A 4% polyacrylamide gel containing 10 μM acrydite modified probe oligos(Integrated DNA Technologies, www.idtdna.com) was cast in a gel cassetteto provide an affinity matrix.

Equimolar amounts of the perfect match and single base mismatch targetswere injected into the affinity gel at 30° C. with an electric field of100 V/cm applied across the gel such that both target molecules would beinitially captured and immobilized at the gel buffer interface. Thetemperature was then increased to 70° C. and a constant electric fieldof 20 V/cm applied to the gel to move the target into the imaging areaof the gel. The temperature was then dropped to 62° C. and a 108 V/cmSCODA focusing field superimposed over an 8 V/cm DC bias as shown inTable 6 was applied to the four source electrodes with a period of 5seconds. The rotation direction of the SCODA focusing field was alteredevery period.

TABLE 6 Focusing plus bias potentials applied. Electrode A Electrode BElectrode C Electrode D Step 1 −108 4 8 4 Step 2 0 −104 8 4 Step 3 0 4−100 4 Step 4 0 4 8 −104

FIG. 26 shows images of concentration taken every 2 minutes. The perfectmatch target was tagged with 6-FAM and shown in green (leading brightspot which focuses to a tight spot), the mismatch target was tagged withCy5 and is shown in red (trailing bright line that is washed from thegel). The camera gain was reduced on the green channel after the firstimage was taken. DNA was injected into the right side of the gel andfocusing plus bias fields were applied. The perfect match target (green)experiences a drift velocity similar to that shown in FIG. 24A and movestowards a central focus location. The more weakly focusing mismatchtarget (red) experiences a velocity field similar to that shown in FIG.24B and is pushed off the edge of the gel by the bias field. Thedirection of application of the applied washing field is indicated bythe white arrow.

This experiment verifies the predictions of FIGS. 24A and 24Bdemonstrating that it is possible to generate two different velocityprofiles for two DNA targets differing by only a single base enablingpreferential focusing of the target with the higher binding energy tothe gel. The images in FIG. 26 confirm that there are two distinctvelocity profiles generated for the two different sequences of targetDNA moving through an affinity matrix under the application of both aSCODA focusing field and a DC bias. A dispersive velocity field isgenerated for the single base mismatch target and a non-dispersivevelocity field is generated for the perfect match target. This exampledemonstrates that it is possible to efficiently enrich for targets withsingle base specificity, and optionally wash a non-desired target off ofan affinity matrix, even if there is a large excess of mismatch targetin the sample.

Example 30: Optimization of Operating Conditions

Different parameters of the SCODA process may be optimized to achievegood sample enrichment at reasonable yields. In embodiments havingimmobilized (and negatively charged) DNA in the gel, a relatively highsalinity running buffer was found to provide both efficient and stablefocusing, as well as minimizing the time required to electrokineticallyinject target DNA from an adjacent sample chamber into the SCODA gel.

Example 31: Optimization of Buffer Salinity

Early attempts of measuring the temperature dependent mobility ofmolecules in an affinity gel as well as the first demonstrations ofsequence specific SCODA were performed in buffers used forelectrophoretic SCODA. These are typically standard electrophoresisbuffers such as tris-borate EDTA (TBE), often diluted 4 to 6 fold toreduce the gel conductivity, enabling the application of high electricfields within thermal limitations imposed by Joule heating, resulting inshorter concentration times. Although it is possible to achieve sequencespecific SCODA based concentration in a 1×TBE buffer (89 mM tris, 89 mMboric acid, 2 mM disodium EDTA), conditions can be further optimized forperformance of sequence specific SCODA due to the relatively lowconcentration of dissociated ions at equilibrium in 1×TBE buffer. A lowconcentration of dissociated ions results in slow hybridizationkinetics, exacerbates ionic depletion associated with immobilizingcharges (oligonucleotide probes) in the gel, and increases the timerequired to electrokinetically inject target DNA into the gel.Calculations using 89 mM tris base and 89 mM boric acid, with a pKa of9.24 for boric acid and a pKa of 8.3 for tris shows a concentration of1.49 mM each of dissociated tris and dissociated boric acid in 1×TBEbuffer.

Example 32: Effect of Salt Concentration on DNA Hybridization

In embodiments used to separate nucleic acids, the presence of positivecounter ions shields the electrostatic repulsion of negatively chargedcomplementary strands of nucleic acid, resulting in increased rates ofhybridization. For example, it is known that increasing theconcentration of Na⁺ ions affects the rate of DNA hybridization in anon-linear manner. The hybridization rate increases by about 10 foldwhen [NaCl] is increased from 10 mM to 1 M of [NaCl], with most of thegain achieved by the time one reaches about 200 mM. At lowconcentrations of positive counter ions, below about 10 mM, the rate ofhybridization is more strongly dependent on salt concentration, roughlyproportional to the cube of the salt concentration. Theoreticalcalculations suggest that the total positive counter ion concentrationof 1×TBE is around 5.5 mM (1.5 mM of dissociated tris, and 4 mM of Na⁺from the disodium EDTA). At this ion concentration it was possible toachieve focusing however the slow hybridization rates resulted in weakfocusing and large final focus spot sizes.

A slow rate of hybridization can lead to weak focusing through anincrease in the phase lag between the changes in electric field andchanges in mobility. Equation (43) describes the SCODA velocity as beingproportional to cos(ϕ), where ϕ represents the phase lag between themobility oscillations and the electric field oscillations. In the caseof ssSCODA a phase lag can result from both a slow thermal response aswell as from slow hybridization kinetics.

This phase lag results in slower focusing times and larger spot sizessince the final spot size is a balance between the inward SCODA-drivendrift, and outward diffusion-driven drift. Faster focusing times arealways desirable as this tends to reduce the overall time to enrich atarget from a complex mixture. A smaller spot size is also desirable asit improves the ability to discriminate between different molecularspecies. As discussed above, when performing SCODA focusing underapplication of a DC bias, the final focus spot will be shifted offcenter by an amount that depends on both the mobility of the target andthe speed of focusing, both of which depend on the strength of theinteraction between the target and the gel bound probes. The amount ofseparation required to discriminate between two similar molecules whenfocusing under bias therefore depends on the final focus spot diameter.Smaller spot diameters should improve the ability to discriminatebetween two targets with similar affinity to the gel bound probes.

At the low rates of hybridization achieved with 1×TBE buffer, reliablefocusing was only achievable with probe concentrations near 100 μM.Increasing the salt concentration from around 5 mM to 200 mM through theaddition of NaCl, while keeping the probe concentration at 100 μM hadthe effect of reducing the final focus spot size as shown in FIGS.27A-D. All images in FIGS. 27A-D were taken after a similar amount offocusing time (approximately 5 min), however the increased salinityresulted in increased Joule heating, which required a four foldreduction of field strength to prevent boiling when focusing with 200 mMNaCl. Probe concentrations are 100 μM, 10 μM, 1 μM, and 100 μM,respectively in FIGS. 27A, 27B, 27C, and 27D. The buffer used in FIGS.27A, 27B, and 27C was 1×TB with 0.2 M NaCl. The buffer used in FIG. 27Dwas 1×TBE. Focusing was not reliable at 10 μM and 1 μM probe in 1×TBEand these results are not shown. Under equivalent conditions in thisexample, addition of 200 mM NaCl to the gel also allowed for focusing ofcomplementary targets at 100 fold lower probe concentrations.

Equation (43) states that the focusing speed is proportional to theelectric field strength, so that fact that comparable focusing times areachieved with a four-fold reduction in electric field strength suggeststhat the field normalized focusing speed is considerably faster underhigh salinity conditions.

Although the total time for focusing was not reduced by the addition of200 mM NaCl, focusing at lower electric field strength may be desirablein some embodiments because lower field strength can limit the degree ofnon-specific electrophoretic SCODA that may occur in an affinity matrixin some embodiments. For example, all target nucleic acid molecules willfocus irrespective of their sequence in the affinity gels used forsequence specific SCODA in embodiments where the thermal gradient isestablished by an electric field due to electrophoretic SCODA. The speedof electrophoretic SCODA focusing increases with electric field, sodecreasing the field strength will have the effect of reducing thenon-specific SCODA focusing speed, allowing one to wash non-target DNAmolecules from the gel more easily by applying a DC bias.

Example 33: Ion Depletion and Bound Charges

The rate at which ions are depleted (or accumulated) at a boundaryincreases as the fraction of charges that are immobile increases. The100 μM probe concentration required to achieve efficient concentrationin 1×TBE results in 2 mM of bound negative charges within the gel when a20 nucleotide probe is used, which is comparable to the total amount ofdissolved negative ions within the gel (around 5.5 mM). This highproportion of bound charge can result in the formation of regions withinthe gel that become depleted of ions when a constant electric field isplaced across the gel as it is during injection and during SCODAfocusing under DC bias.

A high salinity running buffer can therefore help to minimize many ofthe ion depletion problems associated with immobilizing charges in anssSCODA gel by enabling focusing at lower probe concentrations, as wellas reducing the fraction of bound charges by adding additional freecharges.

Example 34: Denaturation of Double Stranded DNA

Target DNA will not interact with the gel immobilized probes unless itis single stranded. The simplest method for generating single strandedDNA from double stranded DNA is to boil samples prior to injection. Onepotential problem with this method is that samples can re-anneal priorto injection reducing the yield of the process, as the re-annealeddouble stranded targets will not interact with the probes and can bewashed off of the gel by the bias field. Theoretical calculations showthat the rate of renaturation of a sample will be proportional to theconcentration of denatured single stranded DNA. Provided targetconcentration and sample salinity are both kept low, renaturation of thesample can be minimized.

To measure the effect of target concentration on renaturation andoverall efficiency, fluorescently labeled double stranded PCR ampliconscomplementary to gel bound probes were diluted into a 250 μL volumecontaining about 2 mM NaCl and denatured by boiling for 5 min followedby cooling in an ice bath for 5 min. The sample was then placed in thesample chamber of a gel cassette, injected into a focusing gel andconcentrated to the center of the gel. After concentration was completethe fluorescence of the final focus spot was measured, and compared tothe fluorescence of the same quantity of target that was manuallypipetted into the center of an empty gel cassette. This experiment wasperformed with 100 ng (2×10¹¹ copies) and 10 ng (2×10¹⁰ copies) ofdouble stranded PCR amplicons. The 100 ng sample resulted in a yield of40% and the 10 ng sample resulted in a yield of 80%. This exampleconfirms that lower sample DNA concentration will result in higheryields.

Example 35: Phase Lag Induced Rotation

As discussed above, in embodiments in which there is a phase lag betweenthe electric field oscillations and the mobility varying oscillations, arotational component will be added to the velocity of molecules movingthrough the affinity matrix. An example of this problem is shown in FIG.28. The targets shown in FIG. 28 focus weakly under SCODA fields andwhen a small bias is applied to wash them from the gel; the wash fieldand the rotational velocity induced by the SCODA fields sum to zero nearthe bottom left corner of the gel. This results in long wash times, andin extreme cases weak trapping of the contaminant fragments. Thedirection of rotation of the electric field used to produce SCODAfocusing is indicated by arrow 34. The direction of the applied washingforce is indicated by arrow 36.

To overcome this problem the direction of the field rotation can bealtered periodically. In other examples described herein, the directionof the field rotation was altered every period. This results in muchcleaner washing and focusing with minimal dead zones. This scheme wasapplied during focus and wash demonstrations described above and shownin FIG. 26, an example in which the mismatched target was cleanly washedfrom the gel without rotation. Under these conditions there is a reducedSCODA focusing velocity due to the phase lag, but there is not anadditional rotational component of the SCODA velocity.

Example 36: Effect of Secondary Structure

Secondary structure in the target DNA will decrease the rate ofhybridization of the target to the immobilized probes. This will havethe effect of reducing the focusing speed by increasing the phase lagdescribed in equation (43). The amount by which secondary structuredecreases the hybridization rate depends on the details of the secondarystructure. With a simple hairpin for example, both the length of thestem and the loop affect the hybridization rate. For most practicalapplications of sequence specific SCODA, where one desires to enrich fora target molecule differing by a single base from contaminatingbackground DNA, both target and background will have similar secondarystructure. In this case the ability to discriminate between target andbackground will not be affected, only the overall process time. Byincreasing the immobilized probe concentration and the electric fieldrotation period one can compensate for the reduced hybridization rate.

There are potentially cases where secondary structure can have an impacton the ability to discriminate a target molecule from backgroundmolecules. It is possible for a single base difference between targetand background to affect the secondary structure in such a way thatbackground DNA has reduced secondary structure and increasedhybridization rates compared to the target, and is the basis for singlestranded conformation polymorphism (SSCP) mutation analysis. This effecthas the potential to both reduce or enhance the ability to successfullyenrich for target DNA, and care must be taken when designing target andprobe sequences to minimize the effects of secondary structure. Once atarget molecule has been chosen, the probe position can be moved aroundthe mutation site. The length of the probe molecule can be adjusted. Insome cases, oligonucleotides can be hybridized to sequences flanking theregion where the probe anneals to further suppress secondary structure.

Example 37: Quantitation of Sequence Specific SCODA Performance

The length dependence of the final focus location while focusing underDC bias was measured and shown to be independent of length for fragmentsranging from 200 nt to 1000 nt in length; an important result, whichimplies that ssSCODA is capable of distinguishing nucleic acid targetsby sequence alone without the need for ensuring that all targets are ofa similar length. Measurements confirmed the ability to enrich fortarget sequences while rejecting contaminating sequences differing fromthe target by only a single base, and the ability to enrich for targetDNA that differs only by a single methylated cytosine residue withrespect to contaminating background DNA molecules.

Example 38: Length Independence of Focusing

The ability to purify nucleic acids based on sequence alone,irrespective of fragment length, eliminates the need to ensure that alltarget fragments are of similar length prior to enrichment. The theoryof sequence specific SCODA presented above predicts that sequencespecific SCODA enrichment should be independent of target length.However, effects not modeled above may lead to length dependence, andexperiments were therefore performed to confirm the length independenceof sequence specific SCODA.

According to the theory of thermally driven sequence specific SCODAdeveloped above, the final focus location under bias should not dependon the length of the target strands. Length dependence of the finalfocus location enters into this expression through the length dependenceof the unimpeded mobility of the target μ₀. However, since both μ(T_(m))and α are proportional to μ₀, the length dependence will cancel fromthis expression. The final focus location of a target concentrated withthermally driven ssSCODA should therefore not depend on the length ofthe target, even if a bias is present.

There are two potential sources of length dependence in the final focuslocation, not modeled above, which must also be considered:electrophoretic SCODA in embodiments where the temperature gradient isestablished by an electric field, and force based dissociation of probetarget duplexes. DNA targets of sufficient length (>200 nucleotides)have a field dependent mobility in the polyacrylamide gels used forsequence specific SCODA, and will therefore experience a sequenceindependent focusing force when focusing fields are applied to the gel.The total focusing force experienced by a target molecule will thereforebe the sum of the contributions from electrophoretic SCODA and sequencespecific SCODA. Under electrophoretic SCODA, the focusing velocity tendsto increase for longer molecules, while the DC velocity tends todecrease so that under bias the final focus location depends on length.The second potential source of length dependence in the final focuslocation is force based dissociation. The theory of affinity SCODApresented above assumed that probe-target dissociation was drivenexclusively by thermal excitations. However it is possible to dissociatedouble stranded DNA with an applied force. Specifically, an externalelectric field pulling on the charged backbone of the target strand canbe used to dissociate the probe-target duplex. The applied electricfield will tend to reduce the free energy term ΔG in equation (35) by anamount equal to the energy gained by the charged molecule moving throughthe electric field. This force will be proportional to the length of thetarget DNA as there will be more charges present for the electric fieldto pull on for longer target molecules, so for a given electric fieldstrength the rate of dissociation should increase with the length of thetarget.

To measure whether or not these two effects contribute significantly tothe length dependence of the final focus location, two different lengthsof target DNA, each containing a sequence complementary to gelimmobilized probes, were focused under bias and the final focus locationmeasured and compared. The target DNA was created by PCR amplificationof a region of pUC19 that contains a sequence complementary to the probesequence in Table 4. Two reactions were performed with a common forwardprimer, and reverse primers were chosen to generate a 250 bp ampliconand a 1000 bp amplicon. The forward primers were fluorescently labeledwith 6-FAM and Cy5 for the 250 bp and 1000 bp fragments respectively.The targets were injected into an affinity gel and focused to the centerbefore applying a bias field. A bias field of 10 V/cm was superimposedover 120 V/cm focusing fields for 10 min at which point the bias wasincreased to 20 V/cm for an additional 7 min. Images of the gel weretaken every 20 sec, with a 1 sec delay between the 6-FAM channel and theCy5 channel. The field rotation period was 5 sec. Images were postprocessed to determine the focus location of each fragment. FIGS. 29Aand 29B show the focus location versus time for the 250 bp (green) and1000 bp (red) fragments. FIG. 29B is an image of final focus of the twofragments at the end of the experiment.

There is a small difference in final location that can be attributed tothe fact that the two images were not taken at the same phase in theSCODA cycle. This example shows that the final focus position does notdepend on length. Thus, under these operating conditions electrophoreticSCODA focusing is much weaker than affinity SCODA focusing, and thataffinity SCODA is driven largely by thermal dissociation rather thanforce-based dissociation. This result confirms that affinity SCODA iscapable of distinguishing nucleic acid targets by sequence alone withoutthe need for ensuring that all targets are of a similar length.

Example 39: Single Base Mismatch Rejection Ratio

To demonstrate the specificity of ssSCODA with respect to rejection ofsequences differing by a single base, different ratios of synthetic 100nt target DNA containing either a perfect match (PM) or single basemismatch (sbMM) to a gel bound probe, were injected into an affinitygel. SCODA focusing in the presence of DC wash fields was performed toremove the excess sbMM DNA. The PM target sequence was labeled with6-FAM and the sbMM with Cy5; after washing the sbMM target from the gelthe amount of fluorescence at the focus location was quantified for eachdye and compared to a calibration run. For the calibration run,equimolar amounts of 6-FAM labeled PM and Cy5 labeled PM target DNA werefocused to the center of the gel and the fluorescence signal at thefocus location was quantified on each channel. The ratio of the signalCy5 channel to the signal on the 6-FAM channel measured during thiscalibration is therefore the signal ratio when the two dye molecules arepresent in equimolar concentrations. By comparing the fluorescenceratios after washing excess sbMM from the gel to the calibration run itwas possible to determine the amount of sbMM DNA rejected from the gelby washing.

Samples containing target sequences shown in Table 4 were added to thesample chamber and an electric field of 50 V/cm was applied across thesample chamber at 45° C. to inject the sample into a gel containing 10μM of immobilized probe. Once the sample was injected into the gel, theliquid in the sample chamber was replaced with clean buffer and SCODAfocusing was performed with a superimposed DC wash field. A focusingfield of 60 V/cm was combined with a DC wash field of 7 V/cm, the latterapplied in the direction opposite to the injection field. It was foundthat this direction for the wash field led to complete rejection of themismatched target DNA in the shortest amount of time. Table 7 belowshows the amount of DNA injected into the gel for each experiment.

TABLE 7 List of targets run for measuring the rejection ratio ofaffinity SCODA with respect to single base differences. Run Description:Cy5 Labeled Target 6-FAM Labeled Target 1:1 Calibration 10 fmol PM 10fmol PM   100:1 1 pmol sbMM 10 fmol PM  1,000:1 10 pmol sbMM 10 fmol PM10,000:1 100 pmol sbMM 10 fmol PM 100,000:1  1 nmol sbMM 10 fmol PM

After the mismatched target had been washed from the gel, the focusingfields were turned off and the temperature of the gel was reduced to 25°C. prior to taking an image of the gel for quantification. It wasimportant to ensure that all images used for quantification were takenat the same temperature, since Cy5 fluorescence is highly temperaturedependent, with the fluorescence decreasing at higher temperatures. Theratio of fluorescence on the Cy5 and 6-FAM channels were compared to the1:1 calibration run to determine the rejection ratio for each run. FIGS.30A and 30B show the results of these experiments. Four different ratiosof sbMM:PM were injected into a gel and focused under bias to removeexcess sbMM. The PM DNA was tagged with 6-FAM and the sbMM DNA wastagged with Cy5. FIG. 30A shows the fluorescence signal from the finalfocus spot after excess sbMM DNA had been washed from the gel. Thefluorescence signals are normalized to the fluorescence measured on aninitial calibration run where a 1:1 ratio of PM 6-FAM:PMCy5 DNA wasinjected and focused to the center of the gel. FIG. 30B shows therejection ratios calculated by dividing the initial ratio of sbMM:PM bythe final ratio after washing.

It was found that rejection ratios of about 10,000 fold are achievable.However it should be noted that images taken during focusing and wash athigh sbMM:PM ratios suggest that there were sbMM molecules with twodistinct velocity profiles. Most of the mismatch target washed cleanlyoff of the gel while a small amount was captured at the focus. Thesefinal focus spots visible on the Cy5 channel likely consisted of Cy5labeled targets that were incorrectly synthesized with the single basesubstitution error that gave them the PM sequence. The 10,000:1rejection ratio measured here corresponds to estimates ofoligonucleotide synthesis error rates with respect to single basesubstitutions, meaning that the mismatch molecule synthesized by theoligonucleotide manufacturer likely contains approximately 1 part in10,000 perfect match molecules. This implies that the residualfluorescence detected on the Cy5 channel, rather than being unresolvedmismatch may in fact be Cy5 labeled perfect match that has been enrichedfrom the mismatch sample. Consequently the rejection ratio of ssSCODAmay actually be higher than 10,000:1.

Example 40: Mutation Enrichment for Clinically Relevant Mutation

The synthetic oligonucleotides used in the example above were purposelydesigned to maximize the difference in binding energy between theperfect match-probe duplex and the mismatch-probe duplex. The ability ofaffinity SCODA to enrich for biologically relevant sequences has alsobeen demonstrated. In this example, cDNA was isolated from cell linesthat contained either a wild type version of the EZH2 gene or a Y641Nmutant, which has previously been shown to be implicated in B-cell nonHodgkin Lymphoma. 460 bp regions of the EZH2 cDNA that contained themutation site were PCR amplified using fluorescent primers in order togenerate fluorescently-tagged target molecules that could be visualizedduring concentration and washing. The difference in binding energybetween the mutant-probe duplex and the wild type-probe duplex at 60° C.was 2.6 kcal/mol compared to 3.8 kcal/mol for the syntheticoligonucleotides used in the previous examples. This corresponds to amelting temperature difference of 5.2° C. for the mutant compared to thewild type. Table 8 shows the free energy of hybridization and meltingtemperature for the wild type and mutants to the probe sequence.

TABLE 8 Binding energy and melting temperatures of EZH2 targets to thegel bound probe. Target Binding Energy Wild Type −161.9 + 0.4646T T_(m)= 57.1° C. Y641N Mutant −175.2 + 0.4966T T_(m) = 62.3° C.

A 1:1 mixture of the two alleles were mixed together and separated withaffinity SCODA. 30 ng of each target amplicon was added to 300 μl of0.01 mM sequence specific SCODA running buffer. The target solution wasimmersed in a boiling water bath for 5 min then placed in an ice bathfor 5 min prior to loading onto the gel cassette in order to denaturethe double stranded targets. The sample was injected with an injectioncurrent of 4 mA for 7 min at 55° C. Once injected, a focusing field of150 V/cm with a 10 V/cm DC bias was applied at 55° C. for 20 min.

The result of this experiment is shown in FIGS. 31A, 31B, and 31C. Thebehavior of these sequences is qualitatively similar to the higher T_(m)difference sequences shown in the above examples. The wild type(mismatch) nucleic acid is completely washed from the gel (images on theright hand side of the figure) while the mutant (perfect match) isdriven towards the center of the gel (images on the left hand side ofthe figure). In this case the efficiency of focusing was reduced as someof the target reannealed forming double stranded DNA that did notinteract with the gel bound probes.

The lower limit of detection with the optical system used was around 10ng of singly labeled 460 bp DNA.

Example 41: Methylation Enrichment

The ability of affinity SCODA based purification to selectively enrichfor molecules with similar binding energies was demonstrated byenriching for methylated DNA in a mixed population of methylated andunmethylated targets with identical sequence.

Fluorescently tagged PM oligonucleotides having the sequence set out inTable 4 (SEQ ID NO.: 2) were synthesized by IDT with a single methylatedcytosine residue within the capture probe region (residue 50 in the PMsequence of Table 4). DC mobility measurements of both the methylatedand unmethylated PM strands were performed to generate velocity versustemperature curves as described above; this curve is shown in FIG. 32.

Fitting of these curves to equation (36) suggests that the difference inbinding energy is around 0.19 kcal/mol at 69° C., which is about a thirdof the thermal energy. (At 69° C. k_(b)T=0.65 kcal/mol.) The curvefurther suggests that separation of the two targets will be mosteffective at an operating temperature of around 69° C., where the twofragments have the greatest difference in mobility as shown in FIG. 33.In this example, the maximum value of this difference is at 69.5° C.,which is the temperature for maximum separation while performing SCODAfocusing under the application of a DC bias.

This temperature is slightly higher than that used in the aboveexamples, and although it should result in better discrimination, focustimes are longer as the higher temperature limits the maximum electricfield strength one can operate at without boiling the gel.

Initial focusing tests showed that it is possible to separate the twotargets by performing affinity SCODA focusing with a superimposed DCbias. FIG. 34 shows the result of an experiment where equimolar ratiosof methylated and unmethylated targets were injected into a gel, focusedwith a period of 5 sec at a focusing field strength of 75 V/cm and abias of 14 V/cm at 69° C. Methylated targets were labeled with 6-FAM(green, spot on right) and unmethylated targets were labeled with Cy5(red, spot on left). The experiment was repeated with the dyes switched,with identical results.

Achieving enrichment by completely washing the unmethylated target fromthe gel proved to be difficult using the same gel geometry for the aboveexamples, as the gel buffer interface was obscured by the buffer wellspreventing the use of visual feedback to control DC bias fields whileattempting to wash the unmethylated target from the gel. To overcomethis problem gels were cast in two steps: first a gel without probeoligonucleotides was cast in one of the arms of the gel and once thefirst gel had polymerized the remainder of the gel area was filled withgel containing probe oligonucleotides. The gels were cast such that theinterface between the two was visible with the fluorescence imagingsystem. This system allowed for real time adjustments in the biasvoltage so that the unmethylated target would enter the gel withoutimmobilized probes and be quickly washed from the gel, while themethylated target could be retained in the focusing gel. FIGS. 35A-35Dshow the result of this experiment. FIGS. 35A and 35B show the resultsof an initial focus before washing unmethylated target from the gel for10 pmol unmethylated DNA (FIG. 35A) and 0.1 pmol methylated DNA (FIG.35B). FIGS. 35C and 35D show the results of a second focusing conductedafter the unmethylated sequence had been washed from the gel forunmethylated and methylated target, respectively. All images were takenwith the same gain and shutter settings.

In this experiment a 100 fold excess of unmethylated target was injectedinto the gel, focused to the center without any wash fields applied. Thetargets were then focused with a bias field to remove the unmethylatedtarget, and finally focused to the center of the gel again forfluorescence quantification. Fluorescence quantification of these imagesindicates that the enrichment factor was 102 fold with losses of themethylated target during washing of 20%. This experiment was repeatedwith the dye molecules swapped (methylated Cy5 and unmethylated 6-FAM)with similar results.

Example 42: Multiplexed Affinity SCODA

Two different oligonucleotide probes described above, one havingaffinity for EZH2 and one having affinity for pUC, were cast in a gel ata concentration of 10 μM each to provide an affinity matrix containingtwo different immobilized probes. A 100 nucleotide target sequence withaffinity for the EZH2 probe and a theoretical melting temperature of62.3° C. was labeled with Cy5. A 100 nucleotide target sequence withaffinity for the pUC probe and a theoretical melting temperature of70.1° C. was labeled with FAM. The theoretical difference in meltingtemperature between the two target molecules is 7.8° C.

The target molecules were loaded on the affinity gel (FIG. 36A), andfocusing was conducted with the temperature beneath the gel boatmaintained at 55° C. (FIGS. 36B, focusing after two minutes, and 36C,after four minutes). The EZH2 target focused under these conditions(four red spots), while the pUC target focused only weakly under theseconditions (three diffuse green spots visible on the gel). The centralextraction well did not contain buffer during the initial portions ofthis experiment, resulting in the production of four focus spots, ratherthan a single central focus spot. The temperature beneath the gel wasthen increased to 62° C., a temperature increase of 7° C. (FIGS. 36D,focusing two minutes after temperature increase, and 36E, after fourminutes), resulting in the formation of four clear focus spots for thepUC target. The EZH2 target remained focused in four tight spots at thishigher temperature.

The temperature beneath the gel was reduced to 55° C. and buffer wasadded to the central extraction well. Application of SCODA focusingfields at this temperature resulted in the EZH2 target being selectivelyconcentrated into the central extraction well (diffuse red spot visibleat the center of FIGS. 36F, 0.5 minutes, and 36G, 1 minute) while thepUC target remained largely focused in four spots outside the centralextraction well. The temperature beneath the gel was increased to 62°C., a temperature increase of 7° C. Within two minutes, the pUC targethad been focused into the central extraction well (FIG. 36H, diffuse redand green fluorescence visible at the center of the gel).

A second experiment was conducted under similar conditions as the first.After focusing the EZH2 target at 55° C. and the pUC target at 62° C. asdescribed above, a DC washing bias was applied to the gel with thetemperature beneath the gel maintained at 55° C. Under these conditions,the EZH2 target experienced a greater bias velocity than the pUC target.The focus spot for the EZH2 target shifted more quickly after theapplication of the bias field (red spot moving to the right of the gelin FIGS. 36I, 6 minutes after application of bias field, 36J, after 12minutes, and 36K, after 18 minutes). The focus spot for the EZH2 targetwas also shifted a farther distance to the right within the gel. Incontrast, the focus spot for the pUC target shifted more slowly (initialgreen focus spots still largely visible in FIG. 36I after 6 minutes,shifting to the right through FIGS. 36J, 12 minutes, and 36K, 18minutes), and was not shifted as far to the right as the focus spot forthe EZH2 target by the washing bias.

Example 43: Affinity SCODA Yield Vs Purity

Because affinity SCODA relies on repeated interactions between targetand probe to generate a non-dispersive velocity field for targetmolecules, while generating a dispersive field for contaminants (so longas a washing bias is applied), high specificity can be achieved withoutsacrificing yield. If one assumes that the final focus spot is Gaussian,which is justified by calculating the spot size for a radial velocityfield balanced against diffusion, then the spot will extend all the wayout to the edge of the gel. Here diffusion can drive targets off the gelwhere there is no restoring focusing force and an applied DC bias willsweep targets away from the gel where they will be lost. In this mannerthe losses for ssSCODA can scale with the amount of time one applies awash field; however the images used to generate FIGS. 27A-27D indicatethat in that example the focus spot has a full width half maximum (FWHM)of 300 μm and under bias it sits at approximately 1.0 mm from the gelcenter. If it is assumed that there is 10 fmol of target in the focusspot, then the concentration at the edge of the gel where a bias isapplied is 1e⁻³⁵² M; that is, there are essentially zero targetmolecules present at the edges of the gel where they can be lost underDC bias. This implies that the rate at which losses accumulate due to anapplied bias (i.e. washing step) is essentially zero. Although thedesired target may be lost from the system in other ways, for example byadsorbing to the sample well prior to injection, running off the edge ofthe gel during injection, re-annealing before or during focusing (in thecase of double stranded target molecules), or during extraction, all ofthese losses are decoupled from the purity of the purified target.

Where a component (e.g. a power supply, electrode, assembly, device,circuit, etc.) is referred to above, unless otherwise indicated,reference to that component (including a reference to a “means”) shouldbe interpreted as including as equivalents of that component anycomponent which performs the function of the described component (i.e.,that is functionally equivalent), including components which are notstructurally equivalent to the disclosed structure which performs thefunction in the illustrated exemplary embodiments of the invention.

Example 44: Isolation of Unknown Mutations with Affinity SCODA

As noted earlier, affinity SCODA may be used to separate similarmolecules with different binding affinities for the immobilized probe.In certain aspects, by varying a driving field along with amobility-altering field, affinity SCODA can be applied to a sample inorder to focus the second molecule with the lesser affinity within anaffinity matrix. In some embodiments, a washing field may be applied tothe affinity matrix which can completely remove the first molecule withthe higher affinity from the affinity matrix. One or more of the fieldsmay be also be applied to the affinity matrix in order to separateand/or remove additional background molecules having a third bindingaffinity for the immobilized probe where the third binding affinity isless than the binding affinities of the first and second molecules.

In an exemplary embodiment, affinity SCODA may be used to resolvenucleic acids with unknown mutations from a sample including bothmutated and wild type nucleic acids. The affinity matrix can includeimmobilized probes comprising covalently bound oligonucleotides whichare a perfect match to the wild type nucleic acids and, therefore, amismatch to the mutated nucleic acid. The oligonucleotide probes canexhibit an affinity for both the wild type nucleic acid as well asmutant nucleic acids where the mutant comprises a substantially similarsequence to the wild type. A mutation may include, for example, a singlenucleotide polymorphism, a base deletion, or a base insertion. Mutantnucleic acids will exhibit a lesser affinity for the immobilized probesthan that of the wild type nucleic acid that is a perfect match to thebound oligonucleotide.

Prior to affinity SCODA enrichment, a fragment of interest may beamplified by PCR amplification or other known methods. The immobilizedprobes can be configured to correspond in length to the entire PCRamplicon in order to cover all potential mutation sites in a fragment ofinterest.

FIG. 37 is an exemplary graph illustrating mobility within an affinitymatrix as a function of temperature for a wild type nucleic acid (WT), amutant nucleic acid (MU), and background molecules where the wild typeis perfectly matched to the probes immobilized on the affinity matrix.As illustrated, WT and MU become increasingly mobile at differenttemperature ranges based on their affinity for the bound probes. Thisdifference may be exploited to separate the WT and MU.

FIGS. 38A-38C illustrate an exemplary embodiment for isolating mutantnucleic acids from both wild type nucleic acids and backgroundmolecules. FIG. 38A shows an exemplary first separation step wherein ahigh magnitude driving field is applied to the affinity matrix in thedirection of extraction while the mobility-altering field, in thisinstance, a temperature, is applied at a magnitude that results in avelocity for the background molecules that is greater than the velocityof MU which is, in turn, greater than the velocity of WT. Using thevalues shown in FIG. 37, a temperature of approximately 59 degreesCelsius could be applied during this step. As a result of this step, thebackground molecules, WT, and MU may be distributed as shown in FIG.38A.

FIG. 38B shows an exemplary next step where a low magnitude drivingfield is applied in the reverse direction, away from extraction. Theconcurrently applied mobility-altering field is now decreased to atemperature resulting in high velocity for the background molecules andlow to negligible velocity for both MU and WT. Using the values shown inFIG. 37, a temperature of approximately 53 degrees Celsius could beapplied during this step. This step results in a distribution as shownin FIG. 38B where MU has made a net movement toward extraction while thebackground molecules and WT are nearer to the edge of the affinitymatrix as represented by the dashed line.

The steps illustrated in FIGS. 38A and 38B correspond to a single cycleand may be repeated to further separate MU from WT.

FIG. 38C illustrates an exemplary wash step where the washing field isapplied in a direction away from extraction and the mobility-alteringfield is increased to a temperature resulting in a large velocity forthe background molecules, WT, and MU. Using the values shown in FIG. 37,a temperature greater than approximately 65 degrees Celsius could beapplied at the washing step. This washing step results in both thebackground molecules and WT being rejected from the affinity matrixwhile MU remains therein.

The mutant nucleic acids, can then be extracted from the affinity matrixand analyzed by any of the means described above. In certainembodiments, the mutant nucleic acids may be sequenced using the methodsdescribed earlier in order to identify unknown mutations in a nucleicacid fragment of interest which may include tumor suppressor genes suchas TP53 or APC.

In certain embodiments, the oligonucleotide probes may be between 50 and100 bases in length. Without being limited to a particular theory, it isbelieved that velocity of a molecule through an affinity matrix isrelated to both temperature and enthalpy thusly:

$v \propto \frac{1}{1 + e^{\frac{\Delta\;{H{({T - T_{m}})}}}{K_{B}T_{m}^{2}}}}$

It is further believed that larger enthalpy values resulting frominteractions between molecules and longer probes, for example, between30 and 500 bp in length, results in steeper velocity curves as afunction of temperature. Steeper velocity curves can enhance the abilityto separate molecules with slight differences in binding affinity byproviding a greater difference in mobility for a given temperature.

T_(m) for the wild type (WT) and mutant (MU) nucleic acids with longer(50-500 bp) bound oligonucleotide probes may be approximated using, forexample, the methods described in Poland Recursion Relation Generationof Probability Profiles for Specific-Sequence Macromolecules withLong-Range Correlations, Biopolymers 1974 13:1859-1871.

These calculations can be used to generate curves such as thoseillustrated in FIGS. 16, 17, and 37. In certain embodiments, thevelocity of the mutant nucleic acid V_(MU) can be calculated using thefollowing formula:

$V_{Mu} = {\frac{E_{L}*t_{L}}{t}\left( {\mu_{H,{Mu}} - \mu_{L,{Mu}}} \right)}$

Where E_(L) is the magnitude of the low magnitude driving field, t_(L)is the time of low magnitude field application, t is the time of asingle cycle of high magnitude forward and low magnitude reverse fieldapplication (as described above in reference to FIGS. 38A and 38B).μ_(H,Mu) and μ_(L,Mu) represent the mutant nucleic acid's targetmobility at the high field temperature and the low field temperaturerespectively where the high field temperature is the temperature appliedduring the high magnitude forward field application and the low fieldtemperature is the temperature applied during the low magnitude reversefield application. A similar equation can be used to calculate thevelocity of the wild type nucleic acid V_(WT):

$V_{WT} = {\frac{E_{L}*t_{L}}{t}\left( {\mu_{H,{WT}} - \mu_{L,{WT}}} \right)}$

Where μ_(H,WT) and μ_(L,WT) represent the wild type nucleic acid'starget mobility at the high field temperature and the low fieldtemperature respectively where the high field temperature is thetemperature applied during the high magnitude forward field applicationand the low field temperature is the temperature applied during the lowmagnitude reverse field application.

Target mobility as a function of temperature is illustrated in FIGS. 16,17, and 37 and may be determined experimentally or, for example, usingthe calculations described above.

In a preferred embodiment, the difference between V_(MU) and V_(WT) (ΔV)is maximized, representing a maximum spatial separation between themutant and wild type nucleic acids within the constraints of the system.This difference can be maximized by minimizing the difference betweenμ_(L,MU) and μ_(L,WT) and maximizing the difference between μ_(H,MU) andμ_(H,WT). These parameters may be minimized and maximized, respectivelythrough means such as configuring the length of the oligonucleotideprobes and altering the low field temperature and the high fieldtemperature.

The length of time in which the washing field is applied (4) does notaffect ΔV because the wild type and mutant nucleic acids haveapproximately equivalent mobility under the mobility-altering field(temperature) applied during the washing step. However, t_(w) can beoptimized to position the wild type and mutant nucleic acids within theaffinity matrix. In preferred embodiment, a value for t_(w) may bedetermined that results in a positive value for V_(MU) and a negativevalue for V_(WT) indicating that, from initial loading into the affinitymatrix, the mutant nucleic acid has experienced a net movement towardextraction while the wild type nucleic acid has experienced a netmovement away from extraction which may represent rejection of the wildtype from the affinity matrix and, therefore, isolation of the mutantnucleic acid. A value for t_(w) to achieve this result may be calculateusing the following formula:

$t_{W} = \frac{E_{L}*t_{L}}{2E_{W}}$

The above method of isolating mutated nucleic acids from a sampleincluding both mutated and wild type nucleic acids is provided as anillustrative example of a larger application. Those of skill in the artwill recognize that the method may be similarly applied to the isolationof any second molecule from a sample including both the second moleculeand a first molecule through the application of a time-varying drivingfield and a periodically varying, mobility-altering field to an affinitymatrix comprising immobilized probes with a first affinity toward thefirst molecule that is greater than a second affinity for a secondmolecule.

In certain embodiments, multiple, unique immobilized probes may be usedin a single affinity matrix wherein said probes are each configured toexhibit a similar T_(m) value, allowing for the isolation of unknownmutations from multiple, unique target fragments in a single,multiplexed assay.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A method for isolating a second molecule from afirst molecule in a sample, the method comprising: applying atime-varying driving field to the sample in the presence of an affinitymatrix comprising immobilized probes, the probes having a first bindingaffinity for the first molecule that is greater than a second bindingaffinity for the second molecule; applying a periodically varying,mobility-altering field that modifies the first and second bindingaffinities; wherein application of the time-varying driving field andthe periodically varying, mobility-altering field concentrates thesecond molecule within the affinity matrix and separate from the firstmolecule; wherein the sample further comprises a background moleculewith a third binding affinity that is less than the first and secondbinding affinities and wherein application of the time-varying drivingfield and periodically varying mobility-altering field concentrates thesecond molecule within the affinity matrix and separate from the firstmolecule and the background molecule.
 2. The method of claim 1, whereinthe periodically varying mobility-altering field is a temperaturegradient.
 3. The method of claim 2, wherein the time-varying drivingfield comprises an electric field and the magnitude of the electricfield is selected to produce the temperature gradient within theaffinity matrix.
 4. The method of claim 1, wherein the time-varyingdriving field comprises an electric field.
 5. The method of claim 1,wherein the time-varying driving field varies direction with time. 6.The method of claim 5, further comprising: calculating a velocity of thefirst and second molecules in the affinity matrix as a function ofmagnitude of the mobility-altering field; determining a value for themagnitude of the mobility-altering field at which the difference inmobility of the first and second molecules is highest; and varying themagnitude of the mobility altering field around the value while varyingthe direction of the driving field to separate the first and secondmolecules within the affinity matrix.
 7. The method of claim 1, furthercomprising applying a washing field to cause net motion of the first andsecond molecules through the affinity matrix.
 8. The method of claim 1,wherein the first and second molecules comprise nucleic acids comprisingbetween 30 and 5000 bases.
 9. The method of claim 1, wherein the firstand second molecules comprise nucleic acids that differ in sequence byat least one base.
 10. The method of claim 1, wherein the first andsecond molecules comprise nucleic acids and the first molecule isidentical to or shares at least 95% sequence similarity with the secondmolecule.
 11. The method of claim 1, wherein the first and secondmolecules comprise oligonucleotides, and wherein the immobilized probeseach comprise a nucleic acid that is complementary to at least a portionof the first molecule.
 12. The method of claim 11, wherein theimmobilized probes each comprise a nucleic acid comprising between 30and 150 bases.
 13. The method of claim 1, wherein the ratio of thesecond molecule in the sample to the first molecule in the sample is1:1,000 or less.
 14. The method of claim 1, wherein a concentration ofthe second molecule relative to a concentration of the first moleculewithin the affinity matrix has been increased by a factor of at least100:1 over the concentration of the second molecule relative to theconcentration of the first molecule in the sample.
 15. The method ofclaim 1, wherein at least one of the first and second molecules isfluorescently labeled, the method further comprising periodicallymonitoring the location of the fluorescently labeled molecules in theaffinity matrix and adjusting the application of the time-varyingdriving field and the periodically varying, mobility-altering fieldbased on the location of the fluorescently labeled molecule.
 16. Themethod of claim 1, wherein two or more target molecules are present inthe sample and are simultaneously concentrated at different, uniquelocations within the affinity matrix.
 17. A method for isolating asecond molecule from a first molecule in a sample, the methodcomprising: applying a time-varying driving field to the sample in thepresence of an affinity matrix comprising immobilized probes, the probeshaving a first binding affinity for the first molecule that is greaterthan a second binding affinity for the second molecule; applying aperiodically varying, mobility-altering field that modifies the firstand second binding affinities; wherein application of the time-varyingdriving field and the periodically varying, mobility-altering fieldconcentrates the second molecule within the affinity matrix and separatefrom the first molecule; wherein the first molecule is a wild typenucleic acid and the second molecule comprises a mutant nucleic acid.18. A method for isolating a second molecule from a first molecule in asample, the method comprising: applying a time-varying driving field tothe sample in the presence of an affinity matrix comprising immobilizedprobes, the probes having a first binding affinity for the firstmolecule that is greater than a second binding affinity for the secondmolecule; applying a periodically varying, mobility-altering field thatmodifies the first and second binding affinities; wherein application ofthe time-varying driving field and the periodically varying,mobility-altering field concentrates the second molecule within theaffinity matrix and separate from the first molecule; wherein the samplecomprises issue, blood, sputum, sweat, urine, feces, tears, aspirate, ora combination thereof.
 19. A method for isolating a second molecule froma first molecule in a sample, the method comprising: applying atime-varying driving field to the sample in the presence of an affinitymatrix comprising immobilized probes, the probes having a first bindingaffinity for the first molecule that is greater than a second bindingaffinity for the second molecule; applying a periodically varying,mobility-altering field that modifies the first and second bindingaffinities; wherein application of the time-varying driving field andthe periodically varying, mobility-altering field concentrates thesecond molecule within the affinity matrix and separate from the firstmolecule; wherein the affinity matrix comprises two or more differentimmobilized probes configured to work simultaneously and whereinapplication of the time-varying driving field and the periodicallyvarying, mobility-altering field simultaneously concentrates a pluralityof different target molecules from the sample.